Competent cells (Nishimura method)
Solution A. 50 ml LB containing 10 mM MgSO4 (0.25 ml of 2 M) + 0.2 % w/v
glucose (0.28 ml 2 M): take out 0.5 ml for use in second step.
Solution B. 3.6 ml glycerol (or weigh out 4.54 g) + 1.2 g PEG-6000 (same as PEG8000) + 60 l 2 M MgSO4 + LB to final volume 10 ml.
Make up fresh, may need to briefly microwave to dissolve PEG, chill to 0oC before
use.
Inoculate 50 ml Solution A with 0.5 ml E. coli overnight.
Shake 37oC ~2 hr, to mid-log phase (OD600 0.3-0.45).
Chill 0oC 10 min.
Pellet in Chilspin 1500xg (2K rpm) 10 min 4oC.
Resuspend bug pellet gently in 0.5 ml chilled Solution A.
Add 2.5 ml chilled Solution B.
Mix gently.
Divide into 0.1 ml aliquots (yellow tip with end cut off), freeze on dry ice.
Store -70oC. Stable for > 1 year.
Transformation.
Thaw cells on ice.
Add 1 - 10 l DNA.
Incubate 15 - 30 min on ice.
Heat pulse 42oC 1 min.
Chill 1-2 min.
Add 0.9 ml LB, incubate 37oC 1 hr.
Plate out aliquots.
Ref. Nishimura et al (1990) Nuc. Ac. Res. 18:6169.