Difference between revisions of "Team:Aix-Marseille/Experiments/Protocols"
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Revision as of 07:36, 13 June 2016
Protocols
Protocol #1 : Preparation of competent bacteria cells
Everything should be done in sterile conditions and on ice (4°C).
- Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
- While it grows, prepare Tbf1 and Tbf2 buffers.
- Centrifuge cells for 10 minutes at 3500g at 4°C
- Resuspend the pellet slowly in 80mL of Tfb1 buffer.
- Centrifuge 5 min at 3500g at 4°C.
- Resuspend the pellet in 8 mL of Tbf2 buffer.
- Incubate 15 min in ice.
- Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.
Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers
For 200 mL of culture:
Preparation of 80 mL of Tbf1 Buffer:
| KAc 1M | 2.4 mL |
| MnCl2 0.5M | 8 mL |
| KCl 1 M | 8 mL |
| CaCl2 0.1M | 8 mL |
| Gly 80% | 15 mL |
| H2O | 38.6 mL |
Preparation of 8 mL of Tbf2 Buffer:
| NaMOPS 0.2M | 400 µL |
| CaCl2 0.1M | 6 mL |
| KCl 1 M | 8 mL |
| Gly 80% | 1.5 mL |
| KCl 1M | 80 µL |
| H2O | 500 µL |
