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Nadine Zimmer — Thu, 11 Oct 2012 14:37:00 GMT

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=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===
{|border="2"
|+'''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===
{|border="2"
|+'''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

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

Nadine Zimmer: erstellt