Waldminghaus:Back Door/Competent E. coli cells with RbCl
From OpenWetWare
Jump to navigationJump to search
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
Kaliumacetat | 0.15g |
MnCl2 x 4H2O | 0.5g |
1M RbCl | 5ml |
1M CaCl2 | 0.5ml |
87%iges Glycerin | .6ml |
A.dest | 35.9ml |
TFB2
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