Difference between revisions of "Waldminghaus:Back Door/Competent E. coli cells with RbCl"
Latest revision as of 07:37, 11 October 2012
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.
|MnCl2 x 4H2O||0.5g|
|1M NaMOPS (pH7)||2.5ml|
|1M CaCl2 x 2H2O||8.75ml|
adjust pH7 and store at 4°C in dark
- 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
- 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