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Summary: Transformation Protocol: RbCl-method
Media and Solutions
YETM, 500 ml: 2.5 g Yeast Extract
10 g Tryptone
5 g MgSO4·7H2O
Adust pH to 7.5 with KOH. For plates: add 7.5 g of Agar
TFB1, 200 ml: 0.59 g KOAc (30 mM)
2.42 g RbCl (100 mM)
0.29 g CaCl2·2H2O (10 mM)
1.98 g MnCl2·4H2O (50 mM)
30 g Glycerol (15% wt/vol)
Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH). Add dH2O to 200 ml. Filter
sterilize. Store refrigerated at 4°C.
TFB2, 200 ml: 0.42 g MOPS (10 mM)
2.21 g CaCl2-2H2O (75 mM)
0.24 g RbCl (10 mM)
30 g Glycerol (15% wt/vol)
Adjust to pH 6.5 with KOH. Add dH2O to 200 ml. Filter sterilize. Store refrigerated at 4°C.
Preparation of Competent Cells
1. Streak cells from frozen stock onto YETM plate. Incubate overnight at 37°C.
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