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<h1><i>E. coli</i></h1>

<h2 id="january">January</h2>



<h3>2015/01/21</h3>

<!-- Transformaion（プレ培養なし） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of <span class="kinyuu">antiBBa_E1010 on BBa_K1524100</span> to <span class="kinyuu">20</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubate 2ml regent with ampicillin at 37℃ for <span class="kinyuu">20</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->







<h3>2015/01/22</h3>

<!-- Colony PCR 2STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.8</span> μL</td></tr>
	<tr><td><span class="kinyuu">XhoⅠ - RBS - NcoⅠ</span> 10 μM</td><td><span class="kinyuu">0.8</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">8.4</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>68℃</td><td>60 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 2STEP END -->


<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Liquid Culture -->
<p class="nyannyan2">Liquid Culture</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td>LB</td><td>2 μL</td></tr>
</table>
<p class="nyannyan3">Cultured for <span class="kinyuu">16</span> hours.</p>
<!-- Liquid Culture END -->








<h3>2015/01/26</h3>

<!-- Colony PCR 2STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">XhoⅠ - RBS - NcoⅠ</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>68℃</td><td>60 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->



<!-- Colony PCR 2STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">XhoⅠ - RBS - NcoⅠ</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>TAKA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>68℃</td><td>60 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 2STEP END -->


<!-- Colony PCR 2STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Sakurai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K1524100</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>TAKA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>68℃</td><td>60 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 2STEP END -->







<h2 id="march">March</h2>

<h3>2015/03/10</h3>

<!-- Competent Cells -->
<h4>Competent Cells</h4>
<p>Tanaka,Sakurai</p>
<p>BL21 (DE3) pLysS</p>
<ol>
	<li>Thawed original competent cells (BL21 (DE3) pLysS) on ice.</li>
	<li>Added 5 μL of original competent cells to 2 mL of LB.</li>
	<li>Incubated the cells for 16 hrs at 37℃.</li>
	<li>Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.</li>
	<li>Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD<sub>600</sub> reach 0.5.</li>
	<li>Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.</li>
	<li>Removed supernatant and added 75 mL of TB to each tube.</li>
	<li>Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.</li>
	<li>Removed supernatant and added 32 mL of TB.</li>
	<li>Added 32 μL of DMSO 10 times.</li>
	<li>Took 50 μL and froze with liquid nitrogen.</li>
</ol>
<!-- Competent Cells END -->






<h3>2015/03/11</h3>

<!-- PCR 3STEP-->
<h4>PCR</h4>
<p>Sakurai</p>
<p>BBa_R0011</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_R0011</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1.5 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1.5 μL</td></tr>
	<tr><td>KOD - Plus - NEO</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - NEO </td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>32 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>

<p>BBa_0030 - BBa_E1010</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_0030 - BBa_E1010</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1.5 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1.5 μL</td></tr>
	<tr><td>KOD - Plus - NEO</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - NEO</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>32 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>30 cycle</td></tr>
	<tr><td>Cycle 2</td><td>62.6℃</td><td>30 sec</td><td>Annealing</td><td>30 cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td>1 min</td><td>Elongation</td><td>30 cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR Purification -->
<h4>PCR Purification</h4>
<p>Sakurai</p>
<p>BBa_R0011, BBa_0030 - BBa_E1010
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>Purification of PCR products</p>
<!-- PCR Purification END -->

<!-- Digestion -->
<h4>Digestion</h4>
<p>Sakurai</p>
<p>BBa_R0011</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_R0011</td><td>44 μL</td></tr>
	<tr><td>XbaI</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>

<p>BBa_0030 - BBa_E1010</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_0030 - BBa_E1010</td><td>44 μL</td></tr>
	<tr><td>XbaI</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>300 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Sakurai</p>
<p>BBa_R0011, BBa_0030 - BBa_E1010</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>1%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<h4>Gel Extract</h4>
<p>Sakurai</p>
<p>BBa_R0011, BBa_0030 - BBa_E1010
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Sakurai</p>
<p>BBa_R0011, BBa_0030 - BBa_E1010</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>1%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<h2 id="may">May</h2>







<h3>2015/05/13</h3>

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Onoda</p>
<p>pET15b</p>
<ol>
	<li>Added 1 μL of pET15b to 50 μL of thawed competent cells (DH5α) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 50 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Onoda, Sakurai</p>
<p>pET16b</p>
<ol>
	<li>Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5α) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 50 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<!-- Competent Cells -->
<h4>Competent Cells</h4>
<p>Onoda</p>
<p>Rosetta</p>
<ol>
	<li>Thawed original competent cells (Rosetta) on ice.</li>
	<li>Added 5 μL of original competent cells to 2 mL of LB.</li>
	<li>Incubated the cells for 16 hrs at 37℃.</li>
	<li>Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.</li>
	<li>Incubated the cells at 130 rpm for 24 hrs at 20℃, until OD<sub>600</sub> reach 0.5.</li>
	<li>Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.</li>
	<li>Removed supernatant and added 75 mL of TB to each tube.</li>
	<li>Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.</li>
	<li>Removed supernatant and added 32 mL of TB.</li>
	<li>Added 32 μL of DMSO 10 times.</li>
	<li>Took 50 μL and froze with liquid nitrogen.</li>
</ol>
<!-- Competent Cells END -->


<h3>2015/05/27</h3>

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Mimata, Onoda, Nishimura</p>
<p>BBa_E0040</p>
<ol>
	<li>Added 1 μL of BBa_E0040 to thawed competent cells (Rosetta and DH5α) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 200 μL of LB.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>mBBa_R0040</p>
<ol>
	<li>Added 1 μL of mBBa_R0040 to thawed competent cells (Rosetta and DH5α) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 200 μL of LB.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<h3>2015/05/29</h3>

<!-- Mini-prep -->
<h4>Mini-prep</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>BBa_E0040, mBBa_R0040
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br>standard protocol</p>
<!-- Mini-prep END -->

<!-- PCR 2STEP-->
<h4>PCR</h4>
<p>Onoda, Ono</p>
<p>BBa_E0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_E0040</td><td>1 μL</td></tr>
	<tr><td>100UP- EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - FX - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - FX - Neo </td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>mBBa_R0040</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - FX - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - FX - Neo </td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>30 cycles</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td>60 sec</td><td>Annealing / Elongation</td><td>30 cycles</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->





<h3>2015/05/30</h3>

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>BBa_E0040, mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>2%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<h4>Gel Extract</h4>
<p>Onoda, Ono, Nishimura</p>
<p>BBa_E0040, mBBa_R0040
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->


<!-- Digestion -->
<h4>Digestion</h4>
<p>Onoda, Ono, Nishimura</p>
<p>BBa_E0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_E0040</td><td>20 μL</td></tr>
        <tr><td>DW</td><td>5 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>3 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p>mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>mBBa_R0040</td><td>20 μL</td></tr>
        <tr><td>DW</td><td>5 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>3 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p>Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->


<!-- Digestion -->
<h4>Digestion</h4>
<p>Onoda, Ono</p>
<p>pET15b</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>pET15b</td><td>10 μL</td></tr>
        <tr><td>DW</td><td>6 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>pET16b</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>pET16b</td><td>10 μL</td></tr>
        <tr><td>DW</td><td>6 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>pSB1A3</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>pSB1A3</td><td>10 μL</td></tr>
        <tr><td>DW</td><td>6 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>pSB4C5</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>pSB4C5</td><td>2 μL</td></tr>
        <tr><td>DW</td><td>14 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->






<h3>2015/05/31</h3>

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3, pSB4C5</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>1%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<h4>Gel Extract</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Mimata, Onoda, Ono, Nishimura</p>
<p>BBa_E0040, mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>1%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- PCR 2STEP-->
<h4>PCR</h4>
<p>Mimata, Onoda</p>
<p>BBa_E0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_E0040</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>mBBa_R0040</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo 10 x Buffer</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>30 cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td>60 sec</td><td>Annealing / Elongation</td><td>30 cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->




<h2 id="june">June</h2>

<h3>2015/06/10</h3>
<!-- Digestion -->
<h4>Digestion</h4>
<p>Mimata, Onoda</p>
<p>BBa_E0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_E0040</td><td>16 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>mBBa_R0040</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>mBBa_R0040</td><td>16 μL</td></tr>
	<tr><td>SpeⅠ</td><td>1 μL</td></tr>
	<tr><td>EcoRⅠ</td><td>1 μL</td></tr>
	<tr><td>CutSmart Buffer</td><td>2 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p>Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->




<h3>2015/06/16</h3>

<!-- PCR 3STEP-->
<h4>PCR</h4>
<p>Mimata</p>
<p>thanatin fragment for TA cloning</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>TA - F - primer</td><td>1 μL</td></tr>
	<tr><td>TA - R - primer</td><td>1 μL</td></tr>
	<tr><td>KAPA Taq</td><td>25 μL</td></tr>
	<tr><td>DW</td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>180 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td>35 cycle</td></tr>
	<tr><td>Cycle 2</td><td>63℃</td><td>30 sec</td><td>Annealing</td><td>35 cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>10 sec</td><td>Elongation</td><td>35 cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>60 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->




<h3>2015/06/17</h3>

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Mimata</p>
<p>thanatin fragment for TA cloning</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>2%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>


<!-- Electrophoresis END -->


<!-- PCR 2STEP-->
<h4>Annealing Oligos and Elongation</h4>
<p>Ito</p>
<p>thanatin fragment for TA cloning</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>TA - F - primer 1 μM</td><td>1 μL</td></tr>
	<tr><td>TA - R - primer 1 μM</td><td>1 μL</td></tr>
	<tr><td>KAPA Taq</td><td>25 μL</td></tr>
	<tr><td>DW</td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>1 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle1</td><td>95℃ to 23℃</td><td>45 min</td><td>Annealing</td><td>1 cycle</td></tr>
	<tr><td>Cycle 2</td><td>72℃</td><td>1 min</td><td>Elongation</td><td>1 cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->




<h3>2015/06/19</h3>


<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Ito</p>
<p>thanatin fragment for TA cloning</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>2%</td><td>100 V</td><td>30min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<h4>Gel Extract</h4>
<p>Ito</p>
<p>thanatin fragment for TA cloning
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->


<!-- Ligation -->
<h4>Ligation</h4>
<p>Ito</p>
<p> pGEM - T vector / Thanatin fragment for TA cloning</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>pGEM - T vector</td><td>1.7μL</td></tr>
	<tr><td>Thanatin fragment for TA cloning</td><td>0.15μL</td></tr>
	<tr><td>Mighty Mix</td><td>1.85μL</td></tr>
	<tr><td>T4 Ligase</td><td>0.18μL</td></tr>
	<tr><td>DW</td><td>6.12μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10μL</b></td></tr>
</table>
<p>Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>2</td><td>65℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<h3>2015/06/21</h3>

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Ito</p>
<p>pGEM - T vector</p>
<ol>
	<li>Added 1 μL of Thanatin fragment to 50 μL of thawed competent cells (Rosseta/DH5α) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 19 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<h2 id="july">July</h2>

<h3>2015/07/25</h3>

<!-- Transformaion（プレ培養あり） -->
<h4>Transformation</h4>
<p>Onoda</p>
<p>BBa_B0015 on pSB1C3</p>
<ol>
	<li>Added 1 μL of BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 500 μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBC.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Transformaion（プレ培養あり） -->
<h4>Transformation</h4>
<p>Onoda</p>
<p>BBa_R0010 - BBa_B0034 on pSB1C3</p>
<ol>
	<li>Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 500 μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBC.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Transformaion（プレ培養あり） -->
<h4>Transformation</h4>
<p>Onoda</p>
<p>BBa_I0500 - BBa_B0034 on pSB1C3</p>
<ol>
	<li>Added 1 μL of BBa_I0500 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 500 μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBC.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Transformaion（プレ培養なし） -->
<h4>Transformation</h4>
<p>Onoda</p>
<p>BBa_B0034 on pSB1A2</p>
<ol>
	<li>Added 1 μL of BBa_B0034 on pSB1A2 to 50 μL of thawed competent cells (DH5α Turbo) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added 500 μL of LB.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for 16 hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<!-- PCR 3STEP-->
<h4>PCR</h4>
<p>Onoda</p>
<p>BBa_B0015 on pSB1C3</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_B0015 on pSB1C3</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>BBa_R0010 - BBa_B0034 on pSB1C3</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_R0010 - BBa_B0034 on pSB1C3</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>BBa_I0500 - BBa_B0034 on pSB1C3</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_I0500 - BBa_B0034 on pSB1C3</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>BBa_B0034 on pSB1A2</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>BBa_B0034 on pSB1A2</td><td>1 μL</td></tr>
	<tr><td>100UP - EX - F 10 μM</td><td>1 μL</td></tr>
	<tr><td>200DN - PS - R 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p>3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>30 cycle</td></tr>
	<tr><td>Cycle 2</td><td>62.6℃</td><td>30 sec</td><td>Annealing</td><td>30 cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td>60 sec</td><td>Elongation</td><td>30 cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Onoda</p>
<p>BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>2%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<h3>2015/07/26</h3>

<!-- Liquid Culture -->
<h4>Liquid Culture</h4>
<p>Ono</p>
<p>BBa_I0500 - BBa_B0034 on pSB1A2</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td>LB</td><td>2000 μL</td></tr>
	<tr><td>Ampicillin</td><td>2 μL</td></tr>
</table>
<p>Cultured for 15 hours.</p>
<!-- Liquid Culture END -->

<!-- Mini-prep -->
<h4>Mini-prep</h4>
<p>Ito</p>
<p>BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0015 on pSB1C3, BBa_B0034 on pSB1A2
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br>standard protocol</p>
<!-- Mini-prep END -->

<!-- Electrophoresis -->
<h4>Electrophoresis</h4>
<p>Ito</p>
<p>BBa_I0500 - BBa_B0034 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A3, BBa_B0015 on pSB1C3</p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td>2%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<h4>Gel Extract</h4>
<p>Ono</p>
<p>BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2, BBa_B0015 on pSB1C3
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<h3>2015/07/27</h3>

<!-- Mini-prep -->
<h4>Mini-prep</h4>
<p>Ono</p>
<p>BBa_I0500 - BBa_B0034 on pSB1A2
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br>standard protocol</p>
<!-- Mini-prep END -->

<h2 id="august">August</h2>

<p class="nyannyan1">2015/08/04</p>

<!-- Transformaion（プレ培養なし） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">pGEM T vector</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">pGEM T vector</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<p class="nyannyan1">2015/08/05</p>

<!-- Colony PCR 2STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">pGEM T vector</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - F - primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - R - primer
</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5.0</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">20</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span>V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">pET vector</span> / <span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pET vector</span></td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></td><td><span class="kinyuu">3</span> μL</td></tr>
	<tr><td>10 × T4 DNA Ligase Buffer</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>T4 Ligase</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>4℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>2</td><td>65℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Transformaion（プレ培養なし） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E1010 - BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">Thanatin on pET vector</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">Rosetta</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->


<p class="nyannyan1">2015/08/10</p>
<!-- Streaking (Single Colony Isolation) -->
<p class="nyannyan2">Streaking (Single Colony Isolation)</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Mimata, Mitsumoto, Onoda, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_E0400 - BBa_B0015 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Picked the colony with an inoculating loop from the agar plate．</li>
	<li>Draged the loop across on a new agar plate.</li>
	<li>Re-sterilised the loop and drag it across again.</li>
</ol>
<!-- Streaking (Single Colony Isolation) END -->




<p class="nyannyan1">2015/08/11</p>
<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Mimata, Mitsumoto, Onoda, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">Fast protocol</span></p>
<!-- Mini-prep END -->


<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Mimata, Onoda, Sakai, Kusumi</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>23 μL</td></tr>
	<tr><td><span class="kinyuu">Bgl Ⅱ</span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">3.1 Buffer</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Mimata, Onoda, Sakai, Nishimura, Kusumi</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<p class="nyannyan1">2015/08/12</p>
<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Sakai, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Gel Extract Poduct)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<p class="nyannyan1">2015/08/13</p>

<!-- Sequencing -->
<p class="nyannyan2">Sequencing</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Onoda, Nishimura, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Gel Extract Product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">
pSB1C3</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">T7 promoter primer / SP6 promoter primer</span></td><td>1.5 μL</td></tr>
	<tr><td>Ready Reaction Premix</td><td>1 μL</td></tr>
	<tr><td>5 x Sequencing Buffer</td><td>1.5 μL</td></tr>
	<tr><td>DW</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10 μL</b></td></tr>
</table>
<p class="nyannyan3">Sequencing</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>96℃</td><td>10 sec</td><td>Denaturation</td><td></td></tr>
	<tr><td>Cycle 1</td><td>50℃</td><td>5 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>60℃</td><td>240 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Sequencing END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Onoda, Nishimura, Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Sequencing PCR product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">2</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">50</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of DW.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Onoda, Tanaka, Nishimura, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin forward Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Asp - Thanatin reverese Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">20</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ on pET vector
</span></p>
<table class="hyounyannyan">
	<tbody><tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ on pET vector</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</tbody></table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</td></tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</tbody></table>
<!-- Digestion END -->

<p class="nyannyan1">2015/08/14</p>

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">T7 - promoter primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SP6 - promoter primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 66℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">50</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ -  Thanatin - BglⅡ, Thanatin fragment(PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Annealing of Oligonucleotides -->
<p class="nyannyan2">Annealing of Oligonucleotides</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Nishimura, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>34 μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">50</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing of Oligonucleotides</p>
<table>
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu"></span>30 sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing of Oligonucleotides END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin  Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Asp - thanatin Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 66℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR Purification -->
<p class="nyannyan2">PCR Purification</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment from last 2 step PCR</span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>Purification of PCR products</p>
<!-- PCR Purification END -->


<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin frament (TA-primer), Thanatin fragment (BamHⅠ/BglⅡ), Thanatin fragment(PCR Purification) </span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Transformaion（プレ培養なし） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2</span> to <span class="kinyuu">20</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">12</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030</span> to <span class="kinyuu">20</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">12</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->







<p class="nyannyan1">2015/08/15</p>


<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 1 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 1 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeI - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Sakai, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - Thanatin - F - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Tanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>34 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>1 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle1</td><td>95℃ to 23℃</td><td>45 min</td><td>Annealing</td><td><span class="kinyuu">1</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">1</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Annealing and Elongation product), Thanatin fragment(PCR 2STEP product) </span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">BBa_B0031, BBa_B0030, BBa_E0040</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">Standard protocol</span></p>
<!-- Mini-prep END -->







<p class="nyannyan1">2015/08/16</p>



<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeI - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers) into DH5α, nothing (as a negative control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">NdeI - F - primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - R - primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">62.9</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2 into DH5α (as positive control) </span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57.2</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>30 sec</td><td>Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment derived from annealing TA primer (colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Mitsumoto, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu"></span>Thanatin fragment (Mini-prep product)</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - Thanatin - F - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Asp - Thanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10　× PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65.1</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu"></span>Thanatin fragment (Mini-prep product)</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeI - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10　× PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">66.5</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment for TA cloning and last PCR prduct</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">60</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment on pGEM - T vector into DH5α</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">NdeI - F - primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - R - primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">62.9</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment on pGEM - T vector into DH5α</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">T7 promoter primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">SP6 promoter primer</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">51</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2 into DH5α (as positive control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>34 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>1 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle1</td><td>95℃ to 23℃</td><td>60 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>34 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td>30 sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - FX - Neo</td><td>1 μL</td></tr>
	<tr><td>10 × PCR Buffer for KOD - FX - Neo</td><td>25 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>14 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>1 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle1</td><td>95℃ to 23℃</td><td>60 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - FX - NEO</td><td>1 μL</td></tr>
	<tr><td>2 × PCR Buffer for KOD - FX - Neo</td><td>25 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>14 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td>30 sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">25</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">23</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">50</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>1 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃ to 23℃</td><td>60 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END-->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">25</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">23</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">50</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td>30 sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA primers)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->



<p class="nyannyan1">2015/08/17</p>




<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>34 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END-->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KAPA Taq</td><td>25 μL</td></tr>
	<tr><td>DW</td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3"> (Tm value &le; -℃)</p>
<table class="hyounyannyan">Annealing Oligos and Elongation</p>
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">10</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Annealing Oligos and Elongation END-->


<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHI - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHI - Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHI - Asp - Thanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Tanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR Purification -->
<p class="nyannyan2">PCR Purification</p>
<p class="nyannyan4"><span class="kinyuu">Ono, MImata, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>Purification of PCR products</p>
<!-- PCR Purification END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA cloning)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing TA cloning)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65.1</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ </span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing TA cloning)</span></td><td>1 μL</td></tr>
<tr><td><span class="kinyuu">BamHⅠ - Asp - Thanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Tanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">66.5</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">45</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>2 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
        <tr><td><span class="kinyuu">BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>2 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->


<p class="nyannyan1">2015/08/18</p>





<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR Purification -->
<p class="nyannyan2">PCR Purification</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Mimata, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>Purification of PCR products</p>
<!-- PCR Purification END -->

<!-- Annealing Oligos and Elongation -->
<p class="nyannyan2">Annealing Oligos and Elongation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing TA cloning)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">TA - F - primer</span> 10 μM</td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">TA - R - primer</span> 10 μM</td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">TE</span> 0.8 M NaCl</td><td>10 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Annealing Oligos and Elongation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>2 min</td><td>Initialization</td><td></td></tr>
	<tr><td>Step1</td><td>95℃ to 25℃</td><td>20 min</td><td>Annealing</td><td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>

<!-- Annealing Oligos and Elongation END-->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Asp - Thanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Tanatin - R - Neo</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR Purification -->
<p class="nyannyan2">PCR Purification</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Mimata, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (PCR 2STEP product)</span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>Purification of PCR products</p>
<!-- PCR Purification END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>2 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
        <tr><td><span class="kinyuu">BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>2 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment on pGEM T - vector</span> / <span class="kinyuu">Thanatin fragment (derived from annealing TA cloning)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pGEM T - vector</span></td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><span class="kinyuu">Thanatin fragment</span></td><td><span class="kinyuu">3</span> μL</td></tr>
	<tr><td>2 × Ligation Buffer</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>T4 Ligase</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>4℃</td><td>6 hour</td><td>Ligation</td></tr>
	<tr><td>2</td><td>65℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->




<p class="nyannyan1">2015/08/19</p>



<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - F - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - R - primer</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (derived from annealing)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (derived from annealing)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - F </span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ  - Tanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>94℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">60</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ (PCR 3STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 3STEP product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">2</span> μL of NaOAc, 1 μL of glycogen and <span class="kinyuu">50</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 30 min.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ, BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Ethanol Precipitation -->

<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ (PCR 2STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 2STEP product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">2</span> μL of NaOAc, 1 μL of glycogen,  <span class="kinyuu">7</span> μL of DW and <span class="kinyuu">50</span> μL of 100% ethanol.</li>
	<li>Left it at room temperature for 15 min.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of DW.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Mimata, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K  Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Mimata, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Mimata, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">pET - 15b vector, pET - 16b vector</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pET - 15b vector, pET - 16b vector</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">NdeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Transformaion（プレ培養なし） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment on pGEM - T vector</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">Thanatin fragment on pGEM - T vector</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Spread 300 μL of the culture onto plate with LBA.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">18</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養なし） END -->




<p class="nyannyan1">2015/08/20</p>

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">NdeⅠ - Thanatin - BamHⅠ (digestion product), BamHⅠ - Thanatin - BglⅡ digestion product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)
</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pET - 15b vector (digestion product), pET - 16b vector (digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">pET - 15b vector, pET - 16b vector</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ(PCR 2STEP poduct)
</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP poduct)</span></td><td>9 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>71 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>100 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nisimura, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)</span></td><td>9 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>71 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>100 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">9</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">270</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 220 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan4"><span class="kinyuu">Ito</span></p>
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product, Ethanol Precipitation product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product, Ethanol Precipitation product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<p class="nyannyan1">2015/08/21</p>

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ, BamHⅠ- Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">3</span> μL of NaOAc, 1 μL of glycogen and <span class="kinyuu">90</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 10 min.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (after Ethanol Prescipitation) BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (after Ethanol Precipitation)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3</span> / <span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_K759012 on pSB1C3</span></td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td>10 × T4 DNA Ligase Buffer</td><td><span class="kinyuu">7</span> μL</td></tr>
	<tr><td>T4 DNA Ligase</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">14</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0015 on pSB1C3</span> / <span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td>10 × T4 DNA Ligase Buffer</td><td><span class="kinyuu">7</span> μL</td></tr>
	<tr><td>T4 DNA Ligase</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">14</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->




<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of <span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer</span></td><td>3 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
        <tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->



<p class="nyannyan1">2015/08/22</p>

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - HF</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>24 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">45</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3, nothing (as a negative control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spidroin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ -D - Thanatin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>40 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2 (as Positive Control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3 (Colony PCR product), BBa_B0031 on pSB1A2 (Colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">45</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span> / <span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td><span class="kinyuu">4</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaI - B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">50</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaI - B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<p class="nyannyan1">2015/08/23</p>

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin, nothing (as a negative control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0030 on pSB1A2 (as a positive control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->




<p class="nyannyan1">2015/08/24</p>

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, nothing (as a negative control), BBa_B0030 on pSB1A2 (as a positive control)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->


<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spidroin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BBa_K759012 - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>40 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, Thanatin - BBa_K759012 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - HF</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Liquid Culture -->
<p class="nyannyan2">Liquid Culture</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3 into DH5α</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td>LB</td><td>2000 μL</td></tr>
	<tr><td>Chloramphenicol</td><td>2 μL</td></tr>
</table>
<p class="nyannyan3">Cultured for <span class="kinyuu">16</span> hours.</p>
<!-- Liquid Culture END -->


<p class="nyannyan1">2015/08/25</p>



<!-- Liquid Culture -->
<p class="nyannyan2">Liquid Culture</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3 into DH5α, BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 into DH5α</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td>LB</td><td>2000 μL</td></tr>
	<tr><td>Chloramphenicol</td><td>2 μL</td></tr>
</table>
<p class="nyannyan3">Cultured for <span class="kinyuu">16</span> hours.</p>
<!-- Liquid Culture END -->

<!-- Sequencing -->
<p class="nyannyan2">Sequencing</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">pbad - f2 / 200 βdomain BBa_K759012 - R</span></td><td>1.5 μL</td></tr>
	<tr><td>BigDye Terminator</td><td>1 μL</td></tr>
	<tr><td>5 x Sequencing Buffer</td><td>1.5 μL</td></tr>
	<tr><td>DW</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10 μL</b></td></tr>
</table>
<p class="nyannyan3">Sequencing</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>96℃</td><td>10 sec</td><td>Denaturation</td><td></td></tr>
	<tr><td>Cycle 1</td><td>50℃</td><td>5 sec</td><td>-</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>60℃</td><td>240 sec</td><td>-</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Sequencing END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 220 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of DW.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">55</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 220 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<p class="nyannyan1">2015/08/26</p>

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono、Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">55</span>℃</td><td>10 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">53</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - XS scar - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP, 3STEP products)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">4</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">120</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Sequencing -->
<p class="nyannyan2">Sequencing</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep producrt)</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F / 200DN - PS - R</span></td><td>1.5 μL</td></tr>
	<tr><td>Ready Reaction Premix</td><td>1 μL</td></tr>
	<tr><td>5 x Sequencing Buffer</td><td>1.5 μL</td></tr>
	<tr><td>DW</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10 μL</b></td></tr>
</table>
<p class="nyannyan3">Sequencing</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>96℃</td><td>10 sec</td><td>Denaturation</td><td></td></tr>
	<tr><td>Cycle 1</td><td>50℃</td><td>5 sec</td><td>-</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>60℃</td><td>240 sec</td><td>-</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Sequencing END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Sequencing PCR product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 220 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<p class="nyannyan1">2015/08/27</p>


<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3 (dephosphorylated)</span> / <span class="kinyuu">BamHⅠ - Thanatin - BglⅡ </span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_K759012 on pSB1C3</span></td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td><span class="kinyuu">4</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">1</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3 (not phosphorylated)</span></p>
<table class="hyounyannyan"> 
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_K759012 on pSB1C3</span></td><td><span class="kinyuu">2</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">2</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">6</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3 (phosphorylated)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_K759012 on pSB1C3</span></td><td><span class="kinyuu">2</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">2</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">6</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Ito</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated) or linearized BBa_K759012 on pSB1C3 (phosphorylated)</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5a</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">12</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - BBa_K759012 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">Agsp - BamHⅠ - Spidroin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BBa_K759012 - bunit - R / BglⅡ - D - Thanatin - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>60 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<p class="nyannyan1">2015/08/28</p>

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 220 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">67</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">67</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END --

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>35 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 3STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">67</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">0.1%BSA</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>2 μL</td></tr>
<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<p class="nyannyan1">2015/08/29</p>

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">20</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">600</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ (digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">3</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">90</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 10 min.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 5 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0033, Thanatin - BBa_K759012, BBa_R0010 - Thanatin, BBa_R0040, BBa_E0040</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ,  XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ,  XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - HF</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<p class="nyannyan1">2015/08/30</p>




<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3, Thanatin - BBa_K759012 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Mimata, Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer </span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Mimata, Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Mimata, Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0030 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0030 on pSB1C3</span></td><td>20 μL</td></tr>
        <tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer </span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>70℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<p class="nyannyan1">2015/08/31</p>








<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Toyooka, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Toyooka, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ- Thanatin - F</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Thanatin - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Toyooka, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Toyooka, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">150</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Fujita, Toyooka, Nishimura </span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Ethanol Precipitation product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (Ethanol Precipitation product)</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>4 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Fujita, Toyooka, Nishimura </span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin fragment (Ethanol Precipitation product)</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Thanatin fragment (Ethanol Precipitation product)</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ</span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ</span></td><td>6 μL</td></tr>
	<tr><td><span class="kinyuu">10 × K Buffer</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>60 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>100 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Toyooka, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Digestion product)</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">280</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Nishimura, Toyooka, Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Ethanol Presipitation product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3</span> / <span class="kinyuu">BamHⅠ- Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_K759012 on pSB1C3</span></td><td><span class="kinyuu">95</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ- Thanatin - BglⅡ</span></td><td><span class="kinyuu">0.5</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_K759012 on pSB1C3, BBa_R0010 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, </span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E1010 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_E1010 on pSB1C3</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer </span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E1010 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_E1010 on pSB1C3</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>7 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E1010 on pSB1C3 (Digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">40</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_E1010 on pSB1C3 (Digestion product)</span></span>
	<br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA family</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">HLA, HLZ, BLA, BLZ</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer </span></td><td>3 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, HLZ, BLA, BLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">HLA, HLZ, BLA, BLZ</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 x M buffer </span></td><td>2 μL</td></tr>
	<tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA family XbaⅠ & SpeⅠ (Digestion product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">40</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Ito, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">HLA family XbaⅠ & SpeⅠ (Digestion product)</span></span>
	<br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->






<h2 id="september">September</h2>


<p class="nyannyan1">2015/09/01</p>

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0015, BBa_I0500</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 on pSB1C3, BBa_I0500 on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3(dephosphorylated product), BBa_R0010 - BBa_B0034 on pSB1C3 (Gel extract product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span> / <span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></td><td><span class="kinyuu">9.5</span> μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td><span class="kinyuu">0.5</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 on pSB1C3</span> / <span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0010 - BBa_B0034 on pSB1C3</span></td><td><span class="kinyuu">9.5</span> μL</td></tr>
	<tr><td><span class="kinyuu">BamHⅠ - Thanatin - BglⅡ</span></td><td><span class="kinyuu">0.5</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin - Ag43 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">2</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">60</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 1 hr.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of TE.</li>
</ol>
<!-- Ethanol Precipitation END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Toyooka</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0033 on pSB1C3, </span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of <span class="kinyuu">BBa_B0033 on pSB1C3, BBa_R0040, BBa_I0500 BBa_B0032 on pSB1C3, BBa_I0500 BBa_B0033 on pSB1C3</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, HLZ, BLA, BLZ, BBa_B0030</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">HLA, HLZ, BLA, BLZ, BBa_B0030</span></td><td>10 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 × M Buffer</span></td><td>2 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>6 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>20 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>60℃</td><td>15 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, HLZ, BLA, BLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Sakai, Ito, Kusumi</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">HLA, HLZ, BLA, BLZ</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Sakai, Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0015 on pSB1C3</span> / <span class="kinyuu">HLA, BLA, BLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td><span class="kinyuu">HLA , BLA, BLZ</span></td><td><span class="kinyuu">30</span> μL</td></tr>
	<tr><td>T4 Ligase</td><td><span class="kinyuu">4.5</span> μL</td></tr>
        <tr><td>10 × T4 DNA Ligase Buffer</td><td><span class="kinyuu">5</span> μL</td></tr>
        <tr><td>DW</td><td><span class="kinyuu">0.5</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">50</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Onoda, Nishimura, Fujita, Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">HLZ</span> / <span class="kinyuu">BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">10</span> μL</td></tr>
	<tr><td><span class="kinyuu">HLZ</span></td><td><span class="kinyuu">20</span> μL</td></tr>
	<tr><td>T4 Ligase</td><td><span class="kinyuu">3.5</span> μL</td></tr>
        <tr><td>10 × T4 DNA Ligase Buffer</td><td><span class="kinyuu">5</span> μL</td></tr>
        <tr><td>DW</td><td><span class="kinyuu">1.5</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">50</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->


<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, HLZ, BLA, BLZ </span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">50</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu"></span> BBa_R0010 - BBa_B0034 on pSB1C3 / <span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ (Digestion product) </span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_R0100 - BBa_B0034 on pSB1C3</span></td><td><span class="kinyuu">15</span> μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ - Thanatin - SpeⅠ</span></td><td><span class="kinyuu">1.0</span> μL</td></tr>
	<tr><td>T4 Ligase</td><td><span class="kinyuu">1.6</span> μL</td></tr>
        <tr><td>10 X T4 DNA Ligase Buffer</td><td><span class="kinyuu">2.0</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">20</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Transformaion -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin - SpeⅠ on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1.0</span> μL of <span class="kinyuu">BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin - SpeⅠ on pSB1C3</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">2</span> hours.</li>
</ol>
<!-- Transformaion END -->


<!-- PCR 2STEP-->
<p class="nyannyan2">PCR</p>
<p class="nyannyan4"><span class="kinyuu">Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">ABF-2, BLA, Thanatin, 10 × His - TEV - Thanatin, BLZ, HLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">ABF-2, BLA, Thanatin, 10 × His - TEV - Thanatin, BLZ, HLZ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">EX - F - Universal</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">PS - R</span> 10 μM</td><td>1 μL</td></tr>
	<tr><td>KOD - Plus - Neo</td><td>1 μL</td></tr>
	<tr><td>10 x PCR Buffer for KOD - Plus - Neo </td><td>5 μL</td></tr>
	<tr><td>2 mM dNTPs</td><td>5 μL</td></tr>
	<tr><td>25 mM MgSO<sub>4</sub></td><td>3 μL</td></tr>
	<tr><td>DW</td><td>33 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">2 Step Cycle (Tm value &ge; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>68℃</td><td><span class="kinyuu">30</span> sec</td><td>Annealing / Elongation</td><td><span class="kinyuu">30</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- PCR 2STEP END -->

<p class="nyannyan1">2015/09/02</p>

<!-- Gel Extract -->
<p class="nyannyan2">Gel Extract</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">EcoRⅠ - BBa_B0032 - XbaⅠ, EcoRⅠ - BBa_B0033 - XbaⅠ, EcoRⅠ - BBa_B0034 - XbaⅠ, SpeⅠ - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_I0500 - SpeⅠ</span></span>
	<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
	<br>DNA extraction from gel</p>
<!-- Gel Extract END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, BLA, HLZ, BLZ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">HLA, BLA, HLZ, BLZ</span> to <span class="kinyuu">10</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spindorin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Thanatin - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spindorin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">Ag43 - bunit - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">50</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">50</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->



<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Nisimura, Ono, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ, HLZ, ABF-2</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BLZ, HLZ, ABF-2</span></td><td>30 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>31 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->



<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ, HLZ, ABF-2,</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">2000</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->


<p class="nyannyan1">2015/09/03</p>

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0015 on pSB1C3</span> / <span class="kinyuu">HLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">6</span> μL</td></tr>
	<tr><td><span class="kinyuu">HLZ</span></td><td><span class="kinyuu">8</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">14</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">28</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0015 on pSB1C3</span> / <span class="kinyuu">HLA, BLA, BLZ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">6</span> μL</td></tr>
	<tr><td><span class="kinyuu">HLA, BLA, BLZ</span></td><td><span class="kinyuu">14</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">20</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">40</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Ligation END -->

<!-- Dephosphorylation -->
<p class="nyannyan2">Dephosphorylation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3">BBa_B0015 on pSB1C3</p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_B0015 on pSB1C3</span></td><td><span class="kinyuu">40</span> μL</td></tr>
	<tr><td>Antarctic Phosphatase</td><td><span class="kinyuu">4</span> μL</td></tr>
	<tr><td>Antarctic Phosphatase Buffer</td><td><span class="kinyuu">8</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">28</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">80</span> μL</b></td></tr>
</table>
<p class="nyannyan3">Dephosphorylation</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>30 min</td><td>Dephosphorylation</td></tr>
	<tr><td>2</td><td>65℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Dephosphorylation END -->


<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ - Thanatin - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spindorin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ - D - Thanatin - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">AGSP - BamHⅠ - Spindorin</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">Ag43 - bunit - Rv</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">65</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_B0031 on pSB1A2</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>40 sec</td><td>Elongation</td><td><span class="kinyuu">40</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">50</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">HLA, BLA, HLZ, BLZ</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">40</span> μL of <span class="kinyuu">HLA, BLA, HLZ, BLZ</span> to <span class="kinyuu">1</span> μL of thawed competent cells (<span class="kinyuu">DH5α</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">16</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Ono, Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Onoda</span></p>
<p class="nyannyan3"><span class="kinyuu">EcoRⅠ - BBa_R0010 - SpeⅠ</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">EcoRⅠ - BBa_R0010 - SpeⅠ</span></td><td>28.5 μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ</span></td><td>1.5 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1.5 μL</td></tr>
	<tr><td><span class="kinyuu">10 × H Buffer</span></td><td>3.5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>35 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<p class="nyannyan1">2015/09/04</p>







<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">ABF-2, BLA, Thanatin, 10 × His - TEV - Thanatin, BLZ, HLZ, ABF-2</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">2</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Ono</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - thanatin</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Sequencing -->
<p class="nyannyan2">Sequencing</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">thanatin - Ag43</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Ag43 thanatin</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">BBa_I0500 - Fw, Ag43 - Rv</span></td><td>1.5 μL</td></tr>
	<tr><td>BigDye Terminator</td><td>1 μL</td></tr>
	<tr><td>5 x Sequencing Buffer</td><td>1.5 μL</td></tr>
	<tr><td>DW</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10 μL</b></td></tr>
</table>
<p class="nyannyan3">Sequencing</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>96℃</td><td>10 sec</td><td>Denaturation</td><td></td></tr>
	<tr><td>Cycle 1</td><td>50℃</td><td>5 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>60℃</td><td>240 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Sequencing END -->


<!-- Sequencing -->
<p class="nyannyan2">Sequencing</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span></td><td>1.5 μL</td></tr>
	<tr><td>Ready Reaction Premix</td><td>1 μL</td></tr>
	<tr><td>5 x Sequencing Buffer</td><td>1.5 μL</td></tr>
	<tr><td>DW</td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>10 μL</b></td></tr>
</table>
<p class="nyannyan3">Sequencing</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>96℃</td><td>10 sec</td><td>Denaturation</td><td></td></tr>
	<tr><td>Cycle 1</td><td>50℃</td><td>5 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td>60℃</td><td>240 sec</td><td>-</td><td><span class="kinyuu">25</span> cycle</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Sequencing END -->

<!-- Ethanol Precipitation -->
<p class="nyannyan2">Ethanol Precipitation</p>
<p class="nyannyan4"><span class="kinyuu">Nishimura, Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - thanatin, BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">1</span> μL of NaOAc, 1.5 μL of glycogen and <span class="kinyuu">30</span> μL of 100% ethanol.</li>
	<li>Left it at -80℃ for 10 min.</li>
	<li>Centrifuged at 15,000 rpm for 15 min at 4℃.</li>
	<li>Removed supernatant and added 100 μL of 70% ethanol.</li>
	<li>Centrifuged at 15,000 rpm for 10 min at 4℃.</li>
	<li>Removed supernatant and air-dried at room temperature with light shield.</li>
	<li>Suspended with 10 μL of HiDi.</li>

<!-- Transformaion（プレ培養あり） -->
<p class="nyannyan2">Transformation</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015</span></p>
<ol class="risutonyannyan">
	<li>Added <span class="kinyuu">5</span> μL of <span class="kinyuu">BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015</span> to <span class="kinyuu">50</span> μL of thawed competent cells (<span class="kinyuu">DH5a</span>) on ice.</li>
	<li>Incubated on ice for 30 min.</li>
	<li>Heat-shocked for 30 sec at 42℃.</li>
	<li>Added <span class="kinyuu">200</span> μL of LB.</li>
	<li>Incubated the cells for 2 hrs at 37℃.</li>
	<li>Spread 300 μL of the culture onto plate with LB<span class="kinyuu">C</span>.</li>
	<li>Incubated the plate at 37℃ for <span class="kinyuu">18</span> hours.</li>
</ol>
<!-- Transformaion（プレ培養あり） END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">PstⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 x H Buffer</span></td><td>5 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>38 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td>5 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>5 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>38 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">PstⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 x H Buffer</span></td><td>5 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>

<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLA</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BLA</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">XbaⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">PstⅠ</span></td><td>1 μL</td></tr>

	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>5 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ, HLZ, ABF-2</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BLZ, HLZ, ABF-2</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1 μL</td></tr>
	<tr><td><span class="kinyuu">10 x H Buffer</span></td><td>5 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>23 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>50 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->






<p class="nyannyan1">2015/09/05</p>









<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3</span>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5.0</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57.6</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu"><p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu"100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->

<!-- Mini-prep -->
<p class="nyannyan2">Mini-prep</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3</span>
	<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
	<br><span class="kinyuu">standard protocol</span></p>
<!-- Mini-prep END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span> / <span class="kinyuu">Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix</span></p>
<table class="hyounyannyan">
	<tbody><tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td><span class="kinyuu">2.5</span> μL</td></tr>
	<tr><td><span class="kinyuu">Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix</span></td><td><span class="kinyuu">40</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">42.5</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">85</span> μL</b></td></tr>
</tbody></table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>2</td><td>70℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</tbody></table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span> / <span class="kinyuu">Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015</span></p>
<table class="hyounyannyan">
	<tbody><tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td><span class="kinyuu">2.5</span> μL</td></tr>
	<tr><td><span class="kinyuu">Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015</span></td><td><span class="kinyuu">40</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">42.5</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">85</span> μL</b></td></tr>
</tbody></table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>2</td><td>70℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</tbody></table>
<!-- Ligation END -->

<!-- Ligation -->
<p class="nyannyan2">Ligation</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span> / <span class="kinyuu">EcoRⅠ - codon optimized - matured HD-5 - PstⅠ</span></p>
<table class="hyounyannyan">
	<tbody><tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">pSB1C3</span></td><td><span class="kinyuu">2.5</span> μL</td></tr>
	<tr><td><span class="kinyuu">EcoRⅠ - codon optimized - matured HD-5 - PstⅠ</span></td><td><span class="kinyuu">30</span> μL</td></tr>
	<tr><td>Mighty Mix</td><td><span class="kinyuu">32.5</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">65</span> μL</b></td></tr>
</tbody></table>
<p class="nyannyan3">Ligation</p>
<table class="hyounyannyan">
	<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr>
	<tr><td>2</td><td>70℃</td><td>10 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</tbody></table>
<!-- Ligation END -->



<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
<p class="nyannyan3"><span class="kinyuu">Thanatin on pSB1C3, TEV - Thanatin on pSB1C3</span></p>
<table class="hyounyannyan">
	<tbody><tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
</tbody></table>
<!-- Electrophoresis END -->




<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">PstⅠ</span></td><td>1.5 μL</td></tr>
	<tr><td><span class="kinyuu">SpeⅠ</span></td><td>1.5 μL</td></tr>
	<tr><td><span class="kinyuu">10 x H Buffer</span></td><td>3 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>4 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->

<!-- Digestion -->
<p class="nyannyan2">Digestion</p>
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
<p class="nyannyan3"><span class="kinyuu">Ag43 - Thanatin</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td><span class="kinyuu">Ag43 - Thanatin</span></td><td>20 μL</td></tr>
	<tr><td><span class="kinyuu">BglⅡ</span></td><td>2.0 μL</td></tr>
	<tr><td><span class="kinyuu">CutSmart Buffer</span></td><td>3 μL</td></tr>
        <tr><td><span class="kinyuu">DW</span></td><td>5 μL</td></tr>
	<tr><td><b>Total</b></td><td><b>30 μL</b></td></tr>
</table>
<p class="nyannyan3">Digestion</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr>
	<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr>
	<tr><td>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
</table>
<!-- Digestion END -->





<p class="nyannyan1">2015/09/06</p>







<!-- Colony PCR 3STEP -->
<p class="nyannyan2">Colony PCR</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3</span></p>
<table class="hyounyannyan">
	<tr><th>Reagent</th><th>Volume</th></tr>
	<tr><td>Single Colony</td><td>-</td></tr>
	<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
	<tr><td>KAPA Taq</td><td><span class="kinyuu">5.0</span> μL</td></tr>
	<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
	<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
</table>
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
<table class="hyounyannyan">
	<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr>
	<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr>
	<tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 2</td><td><span class="kinyuu">57.6</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td><span class="kinyuu">35</span> cycle</td></tr>
	<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr>
	<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr>
</table>
<!-- Colony PCR 3STEP END -->

<!-- Electrophoresis -->
<p class="nyannyan2">Electrophoresis</p>
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
<p class="nyannyan3"><span class="kinyuu">BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 (Colony PCR product)</span></p>
<table class="hyounyannyan">
	<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
	<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
</table>
<!-- Electrophoresis END -->


<p style="text-align:left;"><a href="https://2015.igem.org/Team:HokkaidoU_Japan/Notebook">←Notebook Top</a></p>

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