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
SOB
20 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 MgCl 2/MgSO4 per 100 ml SOB
MgCl2/MgSO4
12 g MgSO4
9.5 g MgCl2.
ddHOH to 100 ml.
Filter sterilize.
FSB
REAGENT
AMT/LITER
FINAL CONC.
1M Potassium Acetate pH 7.5
MnCl2.4H20
CaCl2.2H2O
KCl
Hexammine-cobalt Chloride
Glycerol
10 ml
8.91 g
1.47 g
7.46 g
0.80 g
100 ml
10 mM
45 mM
10 mM
100 mM
3 mM
10%
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 7.5
9.82 g of potassium acetate
90 ml ddHOH
pH 7.5 with glacial acetic acid
Adjust volume to 100 ml
Store at -20oC.