Team:Aix-Marseille/Protocols
DH5 competent cells preparation
- The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
- Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
- The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
- Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
- Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
- Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
- Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
- Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
|
|
200 mL in volume
|
Product and molecular weight
|
| Replicate 1
|
0.0537
|
0.036
|
| Replicate 2
|
0.054099999
|
0.0359
|
| Replicate 3
|
0.056299999
|
0.0361
|
| Replicate 4
|
0.056499999
|
0.0361
|
| Arithmetic Mean
|
0.055
|
0.036
|
| Corrected Abs600
|
0.019
|
|
| Reference OD600
|
0.063
|
|
| OD600/Abs600
|
3.294
|
|
