Difference between revisions of "Team:HokkaidoU Japan/Notebook/ecoli"
Line 447: | Line 447: | ||
</table> | </table> | ||
<!-- PCR 2STEP END --> | <!-- PCR 2STEP END --> | ||
+ | |||
+ | <h2 id="june">June</h2> | ||
+ | |||
+ | <h3>2015/06/10</h3> | ||
+ | <!-- Digestion --> | ||
+ | <h4>Digestion</h4> | ||
+ | <p>Mimata, Onoda</p> | ||
+ | <p>GFP</p> | ||
+ | <table> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>GFP</td><td>16 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>mRFP</p> | ||
+ | <table> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>mRFP</td><td>16 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>600 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> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>TA forward primer</td><td>1 μL</td></tr> | ||
+ | <tr><td>TA reverse 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 ≤ 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>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></td><td>72℃</td><td>60 sec</td><td></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> | ||
+ | <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> | ||
+ | |||
+ | →failed | ||
+ | |||
+ | <!-- Electrophoresis END --> | ||
+ | |||
+ | |||
+ | <!-- PCR 2STEP--> | ||
+ | <h4>Annealing Oligos and Elongation</h4> | ||
+ | <p>実験者</p> | ||
+ | <p>thanatin fragment for TA cloning</p> | ||
+ | <table> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>TA-forward primer 1 μM</td><td>1 μL</td></tr> | ||
+ | <tr><td>TA-reverse 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> | ||
+ | <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>実験者</p> | ||
+ | <p>thanatin fragment for TA cloning</p> | ||
+ | <table> | ||
+ | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
+ | <tr><td>2%</td><td>100V</td><td>30min</td><td>2x TBE</td></tr> | ||
+ | </table> | ||
+ | <!-- Electrophoresis END --> | ||
+ | |||
+ | <!-- Gel Extract --> | ||
+ | <h4>Gel Extract</h4> | ||
+ | <p>実験者</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>実験者</p> | ||
+ | <p>thanatin fragment for TA cloning / Tvector pGEM</p> | ||
+ | <table> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>Tvector pGEM</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> | ||
+ | <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>実験者</p> | ||
+ | <p>Tvector pGEM</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 --> | ||
Revision as of 08:26, 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 |
June
2015/06/10
Digestion
Mimata, Onoda
GFP
Reagent | Volume |
---|---|
GFP | 16 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
mRFP
Reagent | Volume |
---|---|
mRFP | 16 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 600 min | Digestion |
2 | 60℃ | 15 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/06/16
PCR
Mimata
thanatin fragment for TA cloning
Reagent | Volume |
---|---|
TA forward primer | 1 μL |
TA reverse primer | 1 μL |
Kapa Taq | 25 μL |
DW | 23 μL |
Total | 50 μL |
3 Step Cycle (Tm value ≤ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 95℃ | 180 sec | Initialization | |
Cycle 1 | 95℃ | 30 sec | Denaturation | 35 cycle |
Cycle 2 | 63℃ | 30 sec | Annealing | 35 cycle |
Cycle 3 | 72℃ | 10 sec | Elongation | 35 cycle |
72℃ | 60 sec | |||
Store | 4℃ | Hold | Store |
2015/06/17
Electrophoresis
Mimata
thanatin fragment for TA cloning
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 A | 30 min | 2x TBE |
Annealing Oligos and Elongation
実験者
thanatin fragment for TA cloning
Reagent | Volume |
---|---|
TA-forward primer 1 μM | 1 μL |
TA-reverse primer 1 μM | 1 μL |
Kapa Taq | 25 μL |
DW | 23 μL |
Total | 50 μL |
Annealing Oligos and Elongation
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 95℃ | 1 min | Initialization | |
Cycle1 | 95℃ to 23℃ | 45 min | Annealing | 1 cycle |
Cycle 2 | 72℃ | 1 min | Elongation | 1 cycle |
Store | 4℃ | Hold | Store |
2015/06/19
Electrophoresis
実験者
thanatin fragment for TA cloning
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100V | 30min | 2x TBE |
Gel Extract
実験者
thanatin fragment for TA cloning
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ligation
実験者
thanatin fragment for TA cloning / Tvector pGEM
Reagent | Volume |
---|---|
Tvector pGEM | 1.7μL |
thanatin fragment for TA cloning | 0.15μL |
Mighty Mix | 1.85μL |
T4 Ligase | 0.18μL |
DW | 6.12μL |
Total | 10μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
2 | 65℃ | 10 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/06/21
Transformation
実験者
Tvector pGEM
- Added 1 μL of thanatin fragment to 50 μL of thawed competent cells (rosseta/DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 19 hours.