Evening before:

1. Set up overnight culture of bacteria (e.g. DH5-alpha) in 100 ml LMM.
2. Autoclave several flasks of 2X YT broth (1 litre per flask). Aim to process at least 2 flasks. Also autoclave 3 litres of 10% glycerol water (381 g of glycerol dissolved in 3 litres of distilled water).
3. Wash 250 ml centrifuge bottles with caps and screwtops in 0.1% SDS (use brush if necessary) then rinse in distilled water at least 6 times and autoclave.

Next day:

1. Innoculate 10 ml of fresh ON culture into each litre of 2X YT and set shaking at 37 C. Save a small aliquot of uninoculated media as a control for measuring the OD later.
2. Put 10% glycerol water in cold room.
3. Measure OD600 three hours later. Stop culture at OD of about 0.7 - 0.8 (after approx. 5 hours).
4. Chill cells on ice for 20 min.
5. Meanwhile pre-chill JA-14 rotor for Beckman centrifuge (if possible).
6. Pour cells into 250 ml centrifuge bottles spin at 5K for 5 min.
7. Pour off supernatant and resuspend cells in cold 10% glycerol water. Combine resuspended, fill bottles to 3/4 of their capacity with 10% glycerol water and centrifuge again.
8. Resuspend cells in approx 45 ml of 10% glycerol water per large container and transfer to 6 sterile 50 ml tubes and spin these in Beckman centifuge refrigerate at 3.7K for 10 min 4°C.
9. Pour off and resuspend in 50 ml of glycerol water for each tube and repeat spin.
10. Pour off and resuspend each in approx. 15 ml of glycerol water. Combine cells into just two 50 ml tubes and spin at 3.7K for 10 min 4°C.
11. Pour off and resuspend cells in 10% glycerol water to give a final volume of about 5 - 7 mls, depending on yield.
12. Aliquot cells using multi-pipette into eppendorf tubes in 50 µl amounts.

*Keep cells, rotors and centrifuge bottles well chilled at all stages of the process.

TRANSFORMATIONS / ELECTROPORATIONS

1. Ethanol precipiatate 10 ul ligation with 1ul 5M NaCl, 1µl 10 mg/ml tRNA and 25 µl ethanol. Spin for 5 min, rinse with 150 ul of 70% ethanol, spin 2 min and reusupend in 10 µl TE.
2. Immediately prior electroporation add 1 - 2 µl ligation sample to 50 µl aliquot of DH5 alpha electrocompetent cells. Keep on ice
3. Set BTX electroporator to 2.5 Kvolts, 129 ohm capacitance.
4. Have a pasteur pipette with ~1 ml SOC broth ready.
5. Add ligation/DH5 alpha mix to curvette* (0.2 cm electrode gap. Curvettes may be re-used by washing thoroughly in distilled water and storing in 70% ethanol.)
6. Electroporate and then add broth immediately to curvette
7. Transfer contents of curvette to eppendorf tube
8. Incubate @ 37°C for 30-60 minutes
9. Plate onto desired medium.

10 x SOC broth for electroporation

Prepare in dH₂O. Filter sterilize, do not autoclave.

Dilute to 1 X in LMM.