PEG competent bugs
1. Pick a single colony and grow over-night starter (in 2-3ml
LB) *sometimes specific bacterial stains contain
antibiotic resistance. If so: grow in appropriate antibiotic!
2. Put into 100ml LB (dilution 1:100) *the
BL21-SI requires LB with no NaCl (LBON) and not LB!
3. Grow till OD600= 0.3-0.4 *If
you forgot to check and OD was over 0.45, you should dilute the bacterial
suspension using LB to 0.1OD and grow again to 0.3 -0.4!
4. Place in appropriate tubes and spin 1000xg for 10
5. Re-suspend in 1/10 of original volume of very cold
TSS buffer (freshly made)
6. Aliquot into eppendorf tubes, freeze immediately on
dry ice, or directly into –80 (less recommended).
7. Store at –70oC. Cells are best for about 4-6 months,
but will probably work ok for a whole year.
10% w/v PEG (3350 or 8000) *make
sure the powder is dry since PEG is very hygroscopic, and efficiency of
competent cells drops with old wet powder. PEGs of other sizes are NOT
suitable for this procedure!
5% v/v DMSO
50 mM MgCl2 (MgCl2 or MgSO4)
Titrate to final pH 6.5 (essential step!)
Filter sterilize (0.2uM filters).