IGEM:IMPERIAL/2009/XL-1 Blue Preparation

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

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 OD_600nm of 0.5-0.6 (1.6-1.9×10⁸cells/ml)
   1. First doubling may take 1 hour but doublings after that should be every 20-30 mins, so check often!
5. When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes
6. Pellet cells in a centrifuge at 4,000g for 15 mins
7. Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH₂O
8. Fill both tubes to about 350mL with ice cold ddH₂O
   1. Make sure the pellet is fully resuspended!
9. Repellet the cells (as before) and again discard the supernatant
10. Resuspend cells again in 10mL of ddH₂O, then fill both tubes up to about 150mL with ice cold ddH₂O
11. Repellet the cells (as before)
    1. While repelleting, fill the dry ice bath and set up Eppendorf tubes (approximately 50) in a rack in the dry ice bath.
12. 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)
13. Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4,000g
14. Pour off supernatant and resuspend pellet in 2mL of 10% glycerol
15. Pipette 50μL aliquots into the Eppendorfs in the dry ice bath
    1. Freeze on dry ice.
    2. Depending on pipetting accuracy, between 50 and 60 aliquots should be made.
    3. Using a repeating pipette makes this process much faster and reduces risk of contamination.
16. Store at -70°C