Difference between revisions of "Team:HokkaidoU Japan/Notebook/ecoli"
| Line 142: | Line 142: | ||
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- 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 (DH5alpha) 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</p> | ||
| + | <p>pET16b</p> | ||
| + | <ol> | ||
| + | <li>Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5alpha) 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 培養時間 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</p> | ||
| + | <p>GFP</p> | ||
| + | <ol> | ||
| + | <li>Added 1 μL of GFP 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</p> | ||
| + | <p>mRFP</p> | ||
| + | <ol> | ||
| + | <li>Added 1 μL of mRFP 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</p> | ||
| + | <p>GFP, mRFP | ||
| + | <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>GFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>GFP</td><td>1 μL</td></tr> | ||
| + | <tr><td>67-F-primer 10 μL</td><td>1 μL</td></tr> | ||
| + | <tr><td>14-R-primer 10 μL</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD FX NEO</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD FX NEO 10x 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>mRFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>mRFP</td><td>1 μL</td></tr> | ||
| + | <tr><td>67-F-primer 10 μL</td><td>1 μL</td></tr> | ||
| + | <tr><td>14-R-primer 10 μL</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD FX NEO</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD FX NEO 10x 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 ≥ 63℃)</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><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</p> | ||
| + | <p>GFP, mRFP</p> | ||
| + | <table> | ||
| + | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| + | <tr><td>2%</td><td>100 A</td><td>30 min</td><td>2x TBE</td></tr> | ||
| + | </table> | ||
| + | <!-- Electrophoresis END --> | ||
| + | |||
| + | <!-- Gel Extract --> | ||
| + | <h4>Gel Extract</h4> | ||
| + | <p>Onoda, Ono</p> | ||
| + | <p>GFP, mRFP | ||
| + | <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</p> | ||
| + | <p>GFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>GFP</td><td>20 μL</td></tr> | ||
| + | <tr><td>DW</td><td>5 μL</td></tr> | ||
| + | <tr><td>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>3 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>30 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>mRFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>mRFP</td><td>20 μL</td></tr> | ||
| + | <tr><td>DW</td><td>5 μL</td></tr> | ||
| + | <tr><td>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>3 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>30 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>Digestion</p> | ||
| + | <table> | ||
| + | <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> | ||
| + | <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>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>2 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>20 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>pET16b</p> | ||
| + | <table> | ||
| + | <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>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>2 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>20 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>pSB1A3</p> | ||
| + | <table> | ||
| + | <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>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>2 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>20 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>pSB4C5</p> | ||
| + | <table> | ||
| + | <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>Spe1</td><td>1 μL</td></tr> | ||
| + | <tr><td>EcoR1</td><td>1 μL</td></tr> | ||
| + | <tr><td>Cut Smart</td><td>2 μL</td></tr> | ||
| + | <tr><td><b>Total</b></td><td><b>20 μL</b></td></tr> | ||
| + | </table> | ||
| + | <p>Digestion</p> | ||
| + | <table> | ||
| + | <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</p> | ||
| + | <p>GFP, RFP, pET15b, pET16b, pSB1A3, pSB4C5</p> | ||
| + | <table> | ||
| + | <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>1/2x TBE</td></tr> | ||
| + | </table> | ||
| + | <!-- Electrophoresis END --> | ||
| + | |||
| + | <!-- Gel Extract --> | ||
| + | <h4>Gel Extract</h4> | ||
| + | <p>Mimata, Onoda, Ono</p> | ||
| + | <p>GFP, RFP, 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</p> | ||
| + | <p>GFP, mRFP</p> | ||
| + | <table> | ||
| + | <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>1/2x TBE</td></tr> | ||
| + | </table> | ||
| + | <!-- Electrophoresis END --> | ||
| + | |||
| + | |||
| + | <!-- PCR 2STEP--> | ||
| + | <h4>PCR</h4> | ||
| + | <p>Mimata, Onoda</p> | ||
| + | <p>GFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>GFP</td><td>1 μL</td></tr> | ||
| + | <tr><td>67-F-primer 10 μM</td><td>1 μL</td></tr> | ||
| + | <tr><td>14-R-primer 10 μM</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD Plus NEO</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD Plus NEO 10x 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>mRFP</p> | ||
| + | <table> | ||
| + | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| + | <tr><td>mRFP</td><td>1 μL</td></tr> | ||
| + | <tr><td>67-F-primer 10 μM</td><td>1 μL</td></tr> | ||
| + | <tr><td>14-R-primer 10 μM</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD Plus NEO</td><td>1 μL</td></tr> | ||
| + | <tr><td>KOD Plus NEO 10x 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 ≥ 63℃)</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><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 --> | ||
| + | |||
<p style="text-align:left;"><a href="https://2015.igem.org/Team:HokkaidoU_Japan/Notebook">←Notebook Top</a></p> | <p style="text-align:left;"><a href="https://2015.igem.org/Team:HokkaidoU_Japan/Notebook">←Notebook Top</a></p> | ||
Revision as of 08:20, 28 August 2015
E. coli
March
2015/03/10
Competent Cells
mami
BL21 (DE3) pLysS
- Thawed original competent cells (BL21 (DE3) pLysS) on ice.
- Added 5 μL of original competent cells to 2 mL of LB.
- Incubated the cells for 16 hrs at 37℃.
- Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
- Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD600 reach 0.5.
- Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 75 mL of TB to each tube.
- Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 32 mL of TB.
- Added 32 μL of DMSO 10 times.
- Took 50 μL and froze with liquid nitrogen.
2015/03/11
PCR
実験者
BBa_R0011
| Reagent | Volume |
|---|---|
| BBa_R0011 | 1 μL |
| 100bpUP-EX-F 10 μM | 1.5 μL |
| 200bpDN-PS-R 10 μM | 1.5 μL |
| KOD Plus NEO | 1 μL |
| KOD Plus NEO 10x Buffer | 5 μL |
| 2 mM dNTPS | 5 μL |
| 25 mM MgSO4 | 3 μL |
| DW | 32 μL |
| Total | 50 μL |
BBa_0030 - BBa_E1010
| Reagent | Volume |
|---|---|
| BBa_0030 - BBa_E1010 | 1 μL |
| 100bpUP-EX-F 10 μM | 1.5 μL |
| 200bpDN-PS-R 10 μM | 1.5 μL |
| KOD Plus NEO | 1 μL |
| KOD Plus NEO 10x Buffer | 5 μL |
| 2 mM dNTPS | 5 μL |
| 25 mM MgSO4 | 3 μL |
| DW | 32 μL |
| Total | 50 μL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 94℃ | 120 sec | Initialization | |
| Cycle 1 | 98℃ | 10 sec | Denaturation | 30 cycle |
| Cycle 2 | 62.6℃ | 30 sec | Annealing | 30 cycle |
| Cycle 3 | 68℃ | 1 min | Elongation | 30 cycle |
| Store | 4℃ | Hold | Store |
PCR Purification
実験者
BBa_R0011, BBa_0030 - BBa_E1010
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products
Digestion
実験者
BBa_R0011
| Reagent | Volume |
|---|---|
| BBa_R0011 | 44 μL |
| XbaI | 1 μL |
| Cut Smart | 5 μL |
| Total | 50 μL |
BBa_0030 - BBa_E1010
| Reagent | Volume |
|---|---|
| BBa_0030 - BBa_E1010 | 44 μL |
| XbaI | 1 μL |
| Cut Smart | 5 μL |
| Total | 50 μL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 300 min | Digestion |
| 2 | 60℃ | 15 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
実験者
BBa_R0011, BBa_0030 - BBa_E1010
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 30 min | 2x TBE |
Gel Extract
実験者
BBa_R0011, BBa_0030 - BBa_E1010
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Electrophoresis
実験者
BBa_R0011, BBa_0030 - BBa_E1010
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 30 min | 2x TBE |
May
2015/05/13
Transformation
Onoda
pET15b
- Added 1 μL of pET15b to 50 μL of thawed competent cells (DH5alpha) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda
pET16b
- Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5alpha) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Competent Cells
Onoda
rosetta
- Thawed original competent cells (rosetta) on ice.
- Added 5 μL of original competent cells to 2 mL of LB.
- Incubated the cells for 16 hrs at 37℃.
- Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
- Incubated the cells at 130 rpm for 培養時間 hrs at 20℃, until OD600 reach 0.5.
- Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 75 mL of TB to each tube.
- Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 32 mL of TB.
- Added 32 μL of DMSO 10 times.
- Took 50 μL and froze with liquid nitrogen.
2015/05/27
Transformation
Mimata, Onoda
GFP
- Added 1 μL of GFP to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Mimata, Onoda, Ono
mRFP
- Added 1 μL of mRFP to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
2015/05/29
Mini-prep
Mimata, Onoda, Ono
GFP, mRFP
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
PCR
Onoda, Ono
GFP
| Reagent | Volume |
|---|---|
| GFP | 1 μL |
| 67-F-primer 10 μL | 1 μL |
| 14-R-primer 10 μL | 1 μL |
| KOD FX NEO | 1 μL |
| KOD FX NEO 10x Buffer | 5 μL |
| 2 mM dNTPS | 5 μL |
| 25 mM MgSO4 | 3 μL |
| DW | 33 μL |
| Total | 50 μL |
mRFP
| Reagent | Volume |
|---|---|
| mRFP | 1 μL |
| 67-F-primer 10 μL | 1 μL |
| 14-R-primer 10 μL | 1 μL |
| KOD FX NEO | 1 μL |
| KOD FX NEO 10x Buffer | 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 | 30 cycles |
| Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 30 cycles |
| Store | 4℃ | Hold | Store |
2015/05/30
Electrophoresis
Mimata, Onoda, Ono
GFP, mRFP
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100 A | 30 min | 2x TBE |
Gel Extract
Onoda, Ono
GFP, mRFP
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Digestion
Onoda, Ono
GFP
| Reagent | Volume |
|---|---|
| GFP | 20 μL |
| DW | 5 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 3 μL |
| Total | 30 μL |
mRFP
| Reagent | Volume |
|---|---|
| mRFP | 20 μL |
| DW | 5 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 3 μL |
| Total | 30 μL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 60℃ | 15 min | Inactivation |
| Store | 4℃ | Hold | Store |
Digestion
Onoda, Ono
pET15b
| Reagent | Volume |
|---|---|
| pET15b | 10 μL |
| DW | 6 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 2 μL |
| Total | 20 μL |
pET16b
| Reagent | Volume |
|---|---|
| pET16b | 10 μL |
| DW | 6 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 2 μL |
| Total | 20 μL |
pSB1A3
| Reagent | Volume |
|---|---|
| pSB1A3 | 10 μL |
| DW | 6 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 2 μL |
| Total | 20 μL |
pSB4C5
| Reagent | Volume |
|---|---|
| pSB4C5 | 2 μL |
| DW | 14 μL |
| Spe1 | 1 μL |
| EcoR1 | 1 μL |
| Cut Smart | 2 μL |
| Total | 20 μL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 60℃ | 15 min | Inactivation |
| Store | 4℃ | Hold | Store |
2015/05/31
Electrophoresis
Mimata, Onoda, Ono
GFP, RFP, pET15b, pET16b, pSB1A3, pSB4C5
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 30 min | 1/2x TBE |
Gel Extract
Mimata, Onoda, Ono
GFP, RFP, pET15b, pET16b, pSB1A3
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Electrophoresis
Mimata, Onoda, Ono
GFP, mRFP
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 30 min | 1/2x TBE |
PCR
Mimata, Onoda
GFP
| Reagent | Volume |
|---|---|
| GFP | 1 μL |
| 67-F-primer 10 μM | 1 μL |
| 14-R-primer 10 μM | 1 μL |
| KOD Plus NEO | 1 μL |
| KOD Plus NEO 10x Buffer | 5 μL |
| 2 mM dNTPS | 5 μL |
| 25 mM MgSO4 | 3 μL |
| DW | 33 μL |
| Total | 50 μL |
mRFP
| Reagent | Volume |
|---|---|
| mRFP | 1 μL |
| 67-F-primer 10 μM | 1 μL |
| 14-R-primer 10 μM | 1 μL |
| KOD Plus NEO | 1 μL |
| KOD Plus NEO 10x Buffer | 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 | 30 cycle |
| Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 30 cycle |
| Store | 4℃ | Hold | Store |
