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

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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

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
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