Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC9

=Preparation of XL1-Blue Electrocompetent Cells=

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

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

Frequently check the culture whilst growing.


 * 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 innoculating 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