Waldminghaus:Back Door/Competent E. coli cells with RbCl

Competent E. coli cells with RbCl

The general protocol is not very different to CaCl competent cells but cells prepared with RbCl show a higher rate of transformation which is specially important for methods as QuickChange mutagenesis etc.

TFB1

for 50 ml

Kaliumacetat	0.15g
MnCl2 x 4H2O	0.5g
1M RbCl	5ml
1M CaCl2	0.5ml
87%iges Glycerin	.6ml
A.dest	35.9ml

TFB2

for 250 ml

1M NaMOPS (pH7)	2.5ml
1M CaCl2 x 2H2O	8.75ml
1M RbCl	2.5ml
87%iges Glycerin	43.75ml
A.dest	192.5ml

adjust pH7 and store at 4°C in dark

Procedure

• grow 100ml E.coli culture to OD₆₀₀=0.5
• put on ice for 10 min
• centrifuge 2x 50 ml for 10 min at 3000 rpm and 4°C
• wash pellets in 10 ml TFB1 (10 min. 3000 rpm 4°C)
• resuspend pellets in 2ml TFB2
• pipett 200µl aliquots in precooled 1.5 ml tubes
• store at -80°C

Transformation

• let cells melt on ice
• add DNA to be transformed
• let stand on ice for 30 min
• heat shock at 37 °C for 45 sec.
• put back on ice
• add 1 ml LB
• shake at 37 °C for 45 min
• plate on selective plates