Difference between revisions of "Team:HokkaidoU Japan/Notebook/ecoli"
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<!-- Competent Cells --> | <!-- Competent Cells --> | ||
<h4>Competent Cells</h4> | <h4>Competent Cells</h4> | ||
| − | <p | + | <p>mami</p> |
<p>BL21 (DE3) pLysS</p> | <p>BL21 (DE3) pLysS</p> | ||
<ol> | <ol> | ||
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<!-- PCR 3STEP--> | <!-- PCR 3STEP--> | ||
<h4>PCR</h4> | <h4>PCR</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011</p> | <p>BBa_R0011</p> | ||
<table> | <table> | ||
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<!-- PCR Purification --> | <!-- PCR Purification --> | ||
<h4>PCR Purification</h4> | <h4>PCR Purification</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011, BBa_0030 - BBa_E1010 | <p>BBa_R0011, BBa_0030 - BBa_E1010 | ||
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd) | <br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd) | ||
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<!-- Digestion --> | <!-- Digestion --> | ||
<h4>Digestion</h4> | <h4>Digestion</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011</p> | <p>BBa_R0011</p> | ||
<table> | <table> | ||
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<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011, BBa_0030 - BBa_E1010</p> | <p>BBa_R0011, BBa_0030 - BBa_E1010</p> | ||
<table> | <table> | ||
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<!-- Gel Extract --> | <!-- Gel Extract --> | ||
<h4>Gel Extract</h4> | <h4>Gel Extract</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011, BBa_0030 - BBa_E1010 | <p>BBa_R0011, BBa_0030 - BBa_E1010 | ||
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd) | <br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd) | ||
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<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
| − | <p | + | <p>実験者</p> |
<p>BBa_R0011, BBa_0030 - BBa_E1010</p> | <p>BBa_R0011, BBa_0030 - BBa_E1010</p> | ||
<table> | <table> | ||
Revision as of 08:12, 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 |
