COMPETENT CELLS PROTOCOL!!!
hhahn at macc.wisc.edu
Fri Sep 16 12:23:05 EST 1994
In article <stauth-160994084243 at darenmac.usgmrl.ksu.edu>,
stauth at crunch.usgmrl.ksu.edu (Darren Stauth) wrote:
> I've been having problems recently in making competent cells with high
> enough efficiency (>100,000,000 cfu/ug) needed to transform TA clones.
> Does anyone have a good protocol for making high efficiency competent
This is a protocol originally adapted from Chung and Miller, NAR 16:3580.
I routinely get efficiencies around 10^8.
- Grow cells to early log phase (0.3-0.6 A600)
- Pellet cells and discard supernatant
- Resuspend in TSB, 1/10 of original culture volume
- Add glycerol to 15% if freezing and store at -70°C
- Use 100 microliters cells to 10 microliters or less of DNA to transform
Medium (LB, 2xYT, etc) containing
10% PEG 3350
5 % DMSO
10 mM MgCl2
10 mM MgSO4
Harry Hahn / hhahn at macc.wisc.edu / har'ry (har'e) v.t. harass; ravage.
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