IGEM:IMPERIAL/2009/XL-1 Blue Preparation
Preparation of E. coli cells for the cloning of Biobricks and construct construction
- 4,000RPM Centrifuge
- Sterile Centrifugation bottles
- 50ml Tubes
- Large Flasks
- Eppendorf Tubes
- P200 Pipette
- 1 litre of LB medium
- 1-2 litres of autoclaved and chilled ddH2O
- 10% glycerol in ddH2O, autoclaved and chilled
- Dry ice bath
Keep Everything Cold where possible - if using a carbon fibre rotor, you may want to put it in a cold room after innoculation.
Set aside an afternoon for this, starting the culture in the morning
Frequently check the culture whilst growing.
- Grow up a culture of E. coli XL1-blue cells overnight
- Add 40mL of overnight culture to 1 litre of LB medium (containing 20μg/ml Tetracycline)
- Test OD immediately after innoculating the litre flask.
- Grow cells while mixing at at least 225rpm until the culture reaches an OD600nm of 0.5-0.6 (1.6-1.9×108cells/ml)
- First doubling may take 1 hour but doublings after that should be every 20-30 mins, so check often!
- When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes
- Pellet cells in a centrifuge at 4,000g for 15 mins
- Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH2O
- Fill both tubes to about 350mL with ice cold ddH2O
- Make sure the pellet is fully resuspended!
- Repellet the cells (as before) and again discard the supernatant
- Resuspend cells again in 10mL of ddH2O, then fill both tubes up to about 150mL with ice cold ddH2O
- Repellet the cells (as before)
- While repelleting, fill the dry ice bath and set up Eppendorf tubes (approximately 50) in a rack in the dry ice bath.
- Pour off the supernatant and resuspend the cells in 20mL of 10% glycerol (resuspend one pellet then transfer the solution to the other bottle and resuspend the second pellet)
- Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4,000g
- Pour off supernatant and resuspend pellet in 2mL of 10% glycerol
- Pipette 50μL aliquots into the Eppendorfs in the dry ice bath
- Freeze on dry ice.
- Depending on pipetting accuracy, between 50 and 60 aliquots should be made.
- Using a repeating pipette makes this process much faster and reduces risk of contamination.
- Store at -70°C