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