Difference between revisions of "Team:Aix-Marseille/Protocols"
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#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.<br> | #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.<br> | ||
#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.<br> | #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.<br> | ||
| − | 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.<br> | + | #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.<br> |
| − | 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.<br> | + | #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.<br> |
| − | Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.<br> | + | #Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.<br> |
| − | Discard supernatant and resuspend each Falcon tube in 20 mL of solution 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.<br> | + | #Discard supernatant and resuspend each Falcon tube in 20 mL of solution 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.<br> |
| − | Discard supernatant and resuspend each Falcon tube in 8 mL of solution 2.<br> | + | #Discard supernatant and resuspend each Falcon tube in 8 mL of solution 2.<br> |
| − | Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.<br> | + | #Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.<br> |
Revision as of 13:27, 13 August 2018
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 solution 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
- Discard supernatant and resuspend each Falcon tube in 8 mL of solution 2.
- Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.