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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 6 large and 4 small centrifuge containers with caps and screwtops in 0.1% SDS (use brush if necessary) then rinse in distilled water and autoclave.
Next day:
1. Innoculate 10 ml of fresh o/n culture into each litre of 2X YT and set shaking at 37oC. 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'.
5. Meanwhile pre-chill GSA and GS3 (largest) in 5th and 6th floor centrifuges (if possible).
6. Pour cells into 6 large and 4 small containers and spin simultaneously at 5K for 5'.
7. Pour off supernatant and resuspend cells in cold 10% glycerol water. Combine resuspended cells from the small containers into the 6 large containers, then spin these 6 in the GS3 rotor at 5K for 5' (aim to fill these to 3/4 of their capacity with 10% glycerol water).
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' 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' 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 50ul amounts.
N.B. Keep cells, rotors and containers well chilled at all stages of the process.
At one stage it was believed that the milliQ water at Ludwig Institute was impure and gave cells with transformation efficiencies around 107/ug. The conductivity of the water was normal. When water for injection ws used 109-1010/ug was obtained.
TRANSFORMATIONS / ELECTROPORATIONS
1. Ethanol precipiatate 10ul ligation with 1ul 5M NaCl, 1ul 10mg/ml tRNA and 25ul ethanol. SPin for 5min, rinse with 150ul of 70% ethanol, spin 2min and reusupend in 10ul TE.
2. Immediately prior electroporation add 1ul- 2ul ligation sample to 50ul aliquot of DH5a electrocompetant cells. Keep on ice
3. Set BIO-RAD GENE PULSER to 2.5 Kvolts, 25uF and capacitance extender to 200 ohms.
4. Have a pasteur pipette with ~1ml soc broth ready.
5. Add ligation/DH5a mix to curvette* (0.2cm electrode 50 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
per 100 ml per 500 ml
MgCl2 6H2O 2.0g 10g
MgSO4 7H2O 4.9g 24.5g
KCl 180 mg 900 mg
glucose 3.6g 18g
Prepare in dH2O. Filter sterilize, do not autoclave.
Dilute to 1 X in LMM.