Team:Washington/COMPETENT CELL STOCKS

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Latest revision as of 07:16, 7 September 2013

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Chemically Competent Cell Stocks – CCMB Transformation  
For BL21*, CJ236, etc. Cell Lines

NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1).

1. Plate 10ul of cells onto an antibiotic free LB/Agar plate.
2. Incubate overnight at 37OC.
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask.
(I inoculate 4 tips that have been streaked down the entire plate)
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. First check at ~2 hours.
Note: Spectrophotometer needs to warm up for 30 minutes before use.
(I usually end up with an OD of 0.5)
5. Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes.
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.
7. Decant supernatant and invert tubes to remove excess culture medium.
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).  (16ml CCMB per 50ml culture)
9. Incubate on ice for 20 minutes.
10. Centrifuge at 2500rpm for 10 minutes at 4 OC.
11. Decant supernatant and invert tubes to remove excess liquid.
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.
(4ml CCMB per culture)
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting.
13. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.

SOB-Mg Growth Medium – 1L
20g Bacto Tryptone
5g Bacto Yeast Extract
10ml 1M NaCl
2.5ml 1M KCl
dH2O to 1L
Autoclave

CCMB – 1L
10ml 1M Potassium Acetate, pH 7.0 (.9814g)
100g Glycerol
11.8g CaCl2.2H2O
4.0g MnCl2.4H2O
2.0g MgCl2.6H2O (.937 g MgCl)
Sterile filter and store at 4 OC.