Difference between revisions of "Team:HokkaidoU Japan/Notebook/lcasei"
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<h2 id="august">August</h2> | <h2 id="august">August</h2> | ||
| + | <p class="nyannyan1">2015/08/11</p> | ||
| + | |||
| + | <!-- Transformaion（プレ培養なし） --> | ||
| + | <p class="nyannyan2">Transformation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto, Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pHIL253</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added <span class="kinyuu">5</span> μL of <span class="kinyuu">pHIL253</span> to <span class="kinyuu"></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 60 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">2015/08/12</p> | ||
| + | |||
| + | <!-- Liquid Culture --> | ||
| + | <p class="nyannyan2">Liquid Culture</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pHIL253</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>Amp</td><td>2 μL</td></tr> | ||
| + | </table> | ||
| + | <p class="nyannyan3">Cultured for <span class="kinyuu">20</span> hours.</p> | ||
| + | <!-- Liquid Culture END --> | ||
| + | |||
| + | <p class="nyannyan1">2015/08/13</p> | ||
| + | |||
| + | <!-- Mini-prep --> | ||
| + | <p class="nyannyan2">Mini-prep</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pHIL253</span> | ||
| + | <br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd) | ||
| + | <br><span class="kinyuu">standard protocol</span></p> | ||
| + | <!-- Mini-prep END --> | ||
| + | |||
| + | <!-- Preparation of Bacteria --> | ||
| + | <p class="nyannyan2">Preparation of Bacteria</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
| + | <li>Cultured overnight.</li> | ||
| + | <li>Measured OD<sub>600</sub> and diluted with MRS medium to set OD<sub>600</sub> to 0.2.</li> | ||
| + | <li>Stand cultured for 1.5 ~ 2 hours until OD<sub>600</sub> is 0.4 ~ 0.5.</li> | ||
| + | <li>Incubated the cells on ice for 10 min.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 1 mL of cooled PEB.</li> | ||
| + | </ol> | ||
| + | <!-- Preparation of Bacteria END --> | ||
| + | |||
| + | <!-- Electroporation --> | ||
| + | <p class="nyannyan2">Electroporation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Prepared the pHIL253 to 300 ng/10 μL (TE pH 8).</li> | ||
| + | <li>Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.</li> | ||
| + | <li>Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulser<sup>TM</sup> Electroporation Cuvettes(0.2 cm gap #1652086).</li> | ||
| + | <li>Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.</li> | ||
| + | <li>Spread on MRS plate (Em 5 μg/mL),(Amp 5 μg/mL).</li> | ||
| + | </ol> | ||
| + | <!-- Electroporation END --> | ||
| + | |||
| + | |||
| + | <p class="nyannyan1">2015/08/19</p> | ||
| + | |||
| + | <!-- Electrophoresis --> | ||
| + | <p class="nyannyan2">Electrophoresis</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pHIL253</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 --> | ||
| + | |||
| + | <!-- PCR 2STEP--> | ||
| + | <p class="nyannyan2">PCR</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ, proCrp4</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">pHIL253</span></td><td>1 μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - F - primer </span> 10 μM</td><td>1 μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">SpeⅠ - Sec signal sequence - 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 ≥ 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">120</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">2015/08/24</p> | ||
| + | <!-- Ligation --> | ||
| + | <p class="nyannyan2">Ligation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">BBa_E0040 on pSB1A2</span> / <span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">BBa_E0040 on pSB1A2</span></td><td><span class="kinyuu">1</span> μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></td><td><span class="kinyuu">2</span> μL</td></tr> | ||
| + | <tr><td>Mighty Mix</td><td><span class="kinyuu">3</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">4</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>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 --> | ||
| + | |||
| + | <!-- Ligation --> | ||
| + | <p class="nyannyan2">Ligation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">BBa_R0040 on pSB1C3</span> / <span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">BBa_R0040 on pSB1C3</span></td><td><span class="kinyuu">1</span> μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></td><td><span class="kinyuu">2</span> μL</td></tr> | ||
| + | <tr><td>Mighty Mix</td><td><span class="kinyuu">3</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">4</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>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 --> | ||
| + | |||
| + | |||
| + | <!-- Ligation --> | ||
| + | <p class="nyannyan2">Ligation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</span> / <span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</span></td><td><span class="kinyuu">1</span> μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</span></td><td><span class="kinyuu">2</span> μL</td></tr> | ||
| + | <tr><td>Mighty Mix</td><td><span class="kinyuu">3</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">4</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>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（プレ培養あり） --> | ||
| + | <p class="nyannyan2">Transformation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_E0040 on pSB1A2</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added <span class="kinyuu">1</span> μL of <span class="kinyuu">pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_E0040 on pSB1A2</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">18</span> hours.</li> | ||
| + | </ol> | ||
| + | <!-- Transformaion（プレ培養あり） END --> | ||
| + | |||
| + | <p class="nyannyan1">2015/08/26</p> | ||
| + | |||
| + | |||
| + | <!-- Preparation of Bacteria --> | ||
| + | <p class="nyannyan2">Preparation of Bacteria</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mimata, Sakai</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
| + | <li>Cultured overnight.</li> | ||
| + | <li>Measured OD<sub>600</sub> and diluted with MRS medium to set OD<sub>600</sub> to 0.2.</li> | ||
| + | <li>Stand cultured for 1.5 ~ 2 hours until OD<sub>600</sub> is 0.4 ~ 0.5.</li> | ||
| + | <li>Incubated the cells on ice for 10 min.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 1 mL of cooled PEB.</li> | ||
| + | </ol> | ||
| + | <!-- Preparation of Bacteria END --> | ||
| + | |||
| + | |||
| + | <p class="nyannyan1">2015/08/27</p> | ||
| + | |||
| + | <!-- Preparation of Bacteria --> | ||
| + | <p class="nyannyan2">Preparation of Bacteria</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mimata, Sakai</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
| + | <li>Cultured overnight.</li> | ||
| + | <li>Measured OD<sub>600</sub> and diluted with MRS medium to set OD<sub>600</sub> to 0.2.</li> | ||
| + | <li>Stand cultured for 1.5 ~ 2 hours until OD<sub>600</sub> is 0.4 ~ 0.5.</li> | ||
| + | <li>Incubated the cells on ice for 10 min.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 1 mL of cooled PEB.</li> | ||
| + | </ol> | ||
| + | <!-- Preparation of Bacteria END --> | ||
| + | |||
| + | <!-- Electroporation --> | ||
| + | <p class="nyannyan2">Electroporation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mimata, Sakai</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Prepared the plasmid to 300 ng/10 μL (TE pH 8).</li> | ||
| + | <li>Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.</li> | ||
| + | <li>Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulser<sup>TM</sup> Electroporation Cuvettes(0.2 cm gap #1652086).</li> | ||
| + | <li>Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.</li> | ||
| + | <li>Spread on MRS plate (Em 5 μg/mL).</li> | ||
| + | </ol> | ||
| + | <!-- Electroporation END --> | ||
| + | |||
| + | <!-- Colony PCR 3STEP --> | ||
| + | <p class="nyannyan2">Colony PCR</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040</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 ≤ 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>10 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>68℃</td><td>120 sec</td><td>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 3STEP END --> | ||
| + | |||
| + | <!-- Colony PCR 3STEP --> | ||
| + | <p class="nyannyan2">Colony PCR</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040</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 - Sec signal sequence reverse 2</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 ≤ 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>10 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>68℃</td><td>120 sec</td><td>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 3STEP END --> | ||
| + | |||
| + | <p class="nyannyan1">2015/08/28</p> | ||
| + | <!-- Electrophoresis --> | ||
| + | <p class="nyannyan2">Electrophoresis</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mitsumoto</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">colony PCR products of oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3(colony PCR products), oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040(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">45</span> min</td><td>1/2 x TBE</td></tr> | ||
| + | </table> | ||
| + | <!-- Electrophoresis END --> | ||
| + | |||
| + | <p class="nyannyan1">2015/08/31</p> | ||
| + | |||
| + | <!-- Electrophoresis --> | ||
| + | <p class="nyannyan2">Electrophoresis</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_0040 on pSB1A2(colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence 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">40</span> min</td><td>1/2 x TBE</td></tr> | ||
| + | </table> | ||
| + | <!-- Electrophoresis END --> | ||
| + | |||
| + | <!-- Dephosphorylation --> | ||
| + | <p class="nyannyan2">Dephosphorylation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3">BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product) </p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">vector</span></td><td><span class="kinyuu">10</span> μL</td></tr> | ||
| + | <tr><td>Antarctic Phosphatase</td><td><span class="kinyuu">1</span> μL</td></tr> | ||
| + | <tr><td>Antarctic Phosphatase Buffer</td><td><span class="kinyuu">2</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">7</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">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 --> | ||
| + | |||
| + | <!-- Electrophoresis --> | ||
| + | <p class="nyannyan2">Electrophoresis</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 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">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)<span class="kinyuu"></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">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</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 - HF</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 --> | ||
| + | |||
| + | <!-- Electrophoresis --> | ||
| + | <p class="nyannyan2">Electrophoresis</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</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">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence<span class="kinyuu"></span></span> | ||
| + | <br>FastGene<sup>TM</sup> Gel 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">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3</span> / <span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu">BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3</span></td><td><span class="kinyuu">3.5</span> μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></td><td><span class="kinyuu">8</span> μL</td></tr> | ||
| + | <tr><td>Mighty Mix</td><td><span class="kinyuu">11.5</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">7</span> μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b><span class="kinyuu">30</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>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 --> | ||
| + | |||
| + | <!-- Ligation --> | ||
| + | <p class="nyannyan2">Ligation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Onoda</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</span> / <span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></p> | ||
| + | <table class="hyounyannyan"> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td><span class="kinyuu"> pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</span></td><td><span class="kinyuu">20</span> μL</td></tr> | ||
| + | <tr><td><span class="kinyuu">oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></td><td><span class="kinyuu">3</span> μL</td></tr> | ||
| + | <tr><td>Mighty Mix</td><td><span class="kinyuu">23</span> μL</td></tr> | ||
| + | <tr><td>DW</td><td><span class="kinyuu">4</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>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">2015/09/01</p> | ||
| + | |||
| + | |||
| + | <!-- Preparation of Bacteria --> | ||
| + | <p class="nyannyan2">Preparation of Bacteria</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mimata</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
| + | <li>Cultured overnight.</li> | ||
| + | <li>Measured OD<sub>600</sub> and diluted with MRS medium to set OD<sub>600</sub> to 0.2.</li> | ||
| + | <li>Stand cultured for 1.5 ~ 2 hours until OD<sub>600</sub> is 0.4 ~ 0.5.</li> | ||
| + | <li>Incubated the cells on ice for 10 min.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 30 mL of cooled PEB.</li> | ||
| + | <li>Centrifuged at 6,000 g for 15 min at 4℃.</li> | ||
| + | <li>Removed supernatant and added 1 mL of cooled PEB.</li> | ||
| + | </ol> | ||
| + | <!-- Preparation of Bacteria END --> | ||
| + | |||
| + | <!-- Electroporation --> | ||
| + | <p class="nyannyan2">Electroporation</p> | ||
| + | <p class="nyannyan4"><span class="kinyuu">Mimata, Sakai</span></p> | ||
| + | <p class="nyannyan3"><span class="kinyuu">AHU1910</span></p> | ||
| + | <ol class="risutonyannyan"> | ||
| + | <li>Prepared the plasmid to 300 ng/10 μL (TE pH 8).</li> | ||
| + | <li>Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.</li> | ||
| + | <li>Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulser<sup>TM</sup> Electroporation Cuvettes(0.2 cm gap #1652086).</li> | ||
| + | <li>Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.</li> | ||
| + | <li>Spread on MRS plate (Em 5 μg/mL).</li> | ||
| + | </ol> | ||
| + | <!-- Electroporation END --> | ||
| + | |||
<h2 id="september">September</h2> | <h2 id="september">September</h2> | ||
Revision as of 18:24, 18 September 2015
L. casei
August
2015/08/11
Transformation
Mitsumoto, Onoda
pHIL253
- Added 5 μL of pHIL253 to μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 60 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
2015/08/12
Liquid Culture
Mitsumoto
pHIL253
| Reagent | Volume |
|---|---|
| Single Colony | - |
| LB | 2000 μL |
| Amp | 2 μL |
Cultured for 20 hours.
2015/08/13
Mini-prep
Mitsumoto
pHIL253
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Preparation of Bacteria
Mitsumoto
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mitsumoto
AHU1910
- Prepared the pHIL253 to 300 ng/10 μL (TE pH 8).
- Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
- Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
- Spread on MRS plate (Em 5 μg/mL),(Amp 5 μg/mL).
2015/08/19
Electrophoresis
Mitsumoto
pHIL253
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100V | 30 min | 1/2 x TBE |
PCR
Mitsumoto
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - SpeⅠ, proCrp4
| Reagent | Volume |
|---|---|
| pHIL253 | 1 μL |
| XbaⅠ - oripAMβ1 - repE - F - primer 10 μM | 1 μL |
| SpeⅠ - Sec signal sequence - R - primer 10 μM | 1 μL |
| KOD - Plus - NEO | 1 μL |
| 10 × PCR Buffer for KOD - Plus - Neo | 5 μL |
| 2 mM dNTPs | 5 μL |
| 25 mM MgSO4 | 3 μL |
| DW | 33 μL |
| Total | 50 μL |
2 Step Cycle (Tm value ≥ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 94℃ | 120 sec | Initialization | |
| Cycle 1 | 98℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 68℃ | 120 sec | Annealing / Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
2015/08/24
Ligation
Onoda
BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| BBa_E0040 on pSB1A2 | 1 μL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - SpeⅠ | 2 μL |
| Mighty Mix | 3 μL |
| DW | 4 μL |
| Total | 10 μL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| BBa_R0040 on pSB1C3 | 1 μL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - SpeⅠ | 2 μL |
| Mighty Mix | 3 μL |
| DW | 4 μL |
| Total | 10 μL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| pSB1C3 XbaⅠ & SpeⅠ (Digestion product) | 1 μL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - SpeⅠ | 2 μL |
| Mighty Mix | 3 μL |
| DW | 4 μL |
| Total | 10 μL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Transformation
Onoda
pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_E0040 on pSB1A2
- Added 1 μL of pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_E0040 on pSB1A2 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 18 hours.
2015/08/26
Preparation of Bacteria
Mimata, Sakai
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
2015/08/27
Preparation of Bacteria
Mimata, Sakai
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mimata, Sakai
AHU1910
- Prepared the plasmid to 300 ng/10 μL (TE pH 8).
- Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
- Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
- Spread on MRS plate (Em 5 μg/mL).
Colony PCR
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 μM | 0.4 μL |
| 200DN - PS - R 10 μM | 0.4 μL |
| KAPA Taq | 5.0 μL |
| DW | 4.2 μL |
| Total | 10 μL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 68℃ | 120 sec | Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
Colony PCR
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 μM | 0.4 μL |
| SpeⅠ - Sec signal sequence reverse 2 10 μM | 0.4 μL |
| KAPA Taq | 5.0 μL |
| DW | 4.2 μL |
| Total | 10 μL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 68℃ | 120 sec | Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
2015/08/28
Electrophoresis
Mitsumoto
colony PCR products of oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3(colony PCR products), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040(colony PCR product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100V | 45 min | 1/2 x TBE |
2015/08/31
Electrophoresis
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_0040 on pSB1A2(colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence on pSB1C3
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Dephosphorylation
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
| Reagent | Volume |
|---|---|
| vector | 10 μL |
| Antarctic Phosphatase | 1 μL |
| Antarctic Phosphatase Buffer | 2 μL |
| DW | 7 μL |
| Total | 20 μL |
Dephosphorylation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 30 min | Dephosphorylation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Gel Extract
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
FastGeneTM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Digestion
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 20 μL |
| XbaⅠ | 1 μL |
| SpeⅠ - HF | 1 μL |
| 10 × M buffer | 3 μL |
| DW | 5 μL |
| Total | 30 μL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 80℃ | 20 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Gel Extract
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
FastGeneTM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ligation
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 | 3.5 μL |
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 8 μL |
| Mighty Mix | 11.5 μL |
| DW | 7 μL |
| Total | 30 μL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| pSB1C3 XbaⅠ & SpeⅠ (Digestion product) | 20 μL |
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 3 μL |
| Mighty Mix | 23 μL |
| DW | 4 μL |
| Total | 50 μL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
2015/09/01
Preparation of Bacteria
Mimata
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mimata, Sakai
AHU1910
- Prepared the plasmid to 300 ng/10 μL (TE pH 8).
- Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
- Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
- Spread on MRS plate (Em 5 μg/mL).
September
