Team:Imperial College/XL1 Blue

{{Imperial/Box1|Preparation of XL1-Blue Electrocompetent cells|

Aims
Preparation of E. coli cells for the cloning of Biobricks and construct construction

Equipment
4,000RPM Centrifuge

Sterile Centrifugation bottles

50ml Tubes

Large Flasks

Eppendorf Tubes

P200 Pipette

Stripettes

Reagents
1 litre of LB medium

Tetracycline

1-2 litres of autoclaved and chilled ddH2O

10% glycerol in ddH2O, autoclaved and chilled

Dry ice bath

Protocol
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

Check the culture while growing frequently


 * 1) Grow up a culture of E. coli XL1-blue cells overnight
 * 2) Add 40mL of overnight culture to 1 litre of LB medium (containing 20μg/ml Tetracycline)
 * 3) Test OD immediately after inoculating the litre flask.
 * 4) 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)
 * 5) *First doubling may take 1 hour but doublings after that should be every 20-30 mins, so check often!
 * 6) When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes
 * 7) Pellet cells in a centrifuge at 4,000g for 15 mins
 * 8) Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH2O
 * 9) Fill both tubes to about 350mL with ice cold ddH2O
 * 10) *Make sure the pellet is fully resuspended!
 * 11) Repellet the cells (as before) and again discard the supernatant
 * 12) Resuspend cells again in 10mL of ddH2O, then fill both tubes up to about 150mL with ice cold ddH2O
 * 13) Repellet the cells (as before)
 * 14) *While repelleting, fill the dry ice bath and set up Eppendorf tubes (approximately 50) in a rack in the dry ice bath
 * 15) 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)
 * 16) Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4,000g
 * 17) Pour off supernatant and resuspend pellet in 2mL of 10% glycerol
 * 18) Pipette 50μL aliquots into the Eppendorfs in the dry ice bath
 * 19) *Freeze on dry ice
 * 20) *Depending on pipetting accuracy, between 50 and 60 aliquots should be made
 * 21) *Using a repeating pipette makes this process much faster and reduces risk of contamination
 * 22) Store at -70°C
 * }}