Super Competent Cell Prep (Frozen Stocks)
Adapted from Maniatis, second edition p. 1.76->1.81.
This prep works well for E. coli strains DH1, DH5, and MM294; it does not work for MC1061. Routinely, this prep should yield at least 5x107 tfm/ug.
To help guarantee success and reproducability, use only the highest quality reagents for this procedure. Also make sure all lab ware is free of detergents because these surfactants inhibit transformation.
This entire protocol should be done asceptically.
1. Streak DH5a from a frozen stock stored at -70oC onto an LB agar plate. Incubate for ~16 hours at 37oC. Cultures which have been passaged continuously in the lab or stored at 5 or 25oC do not yield transformants at as high a frequency and should not be used.
2. Inoculate an overnight culture (10 ml LB) with 1 colony. Inoculate 250 ml SOB ( in a 1 L flask) with 2.5 ml overnight culture. Grow to an OD600=0.6; approx. 3-4 hours. Do not allow the culture to overgrow (exceed 108 cells/ml); cultures approaching the stationary phase do not transform at a high efficiency.
3. Transfer culture to sterile, ice-cold 50 ml P.P. tubes. Store on ice for 10 min.
4. Centrifuge at 4, k/10 min at 4oC, in pre-chilled JS 13.1 rotor.
5. Decant media from the cells. Stand the inverted tubes an a clean paper towel for one minute to allow media to drain away.
6. Resuspend the pellets by gentle vortexing in 15 ml of FSB (per tube.) Store the resuspended cells on ice for 10 minutes.
7. Repeat steps 4 and 5 and resuspend pellets by gentle vortexing, in 4 ml of FSB (per tube.)
8. Add 140 ul DMSO per 4 ml resuspended cells. Mix gently by swirling, and store the suspension on ice for 15 minutes.
9. Add additional 140 ul of DMSO to each suspension. Mix gently by swirling and return the suspensions to an ice bath.
10. Working quickly, dispense 100 ul aliquots into sterile, pre-chilled eppendorf tubes. Close the tubes and snap-freeze the cells by immersing them in N2(l). Immediately store -70oC until needed.
MEDIA AND BUFFERS
SOB20 g Bacto-Tryptone
5 g Yeast Extract
0.5 g NaCl
ddHOH to 1 L
(Agar= 18 g/L; if desired)
Autoclave
Before use add 1 ml of sterile MgCl2/MgSO4 per 100 ml SOB
MgCl2/MgSO412 g MgSO4
9.5 g MgCl2.
ddHOH to 100 ml.
Filter sterilize.
FSB
REAGENTAMT/LITERFINAL CONC.
1M Potassium Acetate pH 7.510 ml10 mM
MnCl2.4H208.91 g 45 mM
CaCl2.2H2O1.47 g10 mM
KCl7.46 g100 mM
Hexammine-cobalt Chloride0.80 g 3 mM
Glycerol100 ml10%
Mix the components with 800 ml ddHOH.
Adjuste to pH 6.4 with 0.1 N HCl (if you overshoot, do not back titrate with base. Discard the solution. and start over).
Adjust vol. to 1 L
Filter sterilize and store at -20oC.
1 M Potassium acetate pH 7.59.82 g of potassium acetate
90 ml ddHOH
pH 7.5 with glacial acetic acid
Adjust volume to 100 ml
Store at -20oC.