peg_competent bugs (procedure)

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 min (4°C)
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.

TSS buffer
LB
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).