| Potassium acetate (1 m, pH 7.0)
|
10 mL |
|
| CaCl2·2H2O
|
11.8 g |
|
| MnCl2·4H2O
|
4.0 g |
|
| MgCl2·6H2O
|
2.0 g |
|
| Glycerol |
100 mL |
10% (v/v) |
| Detergents and organic contaminants are strong inhibitors of transformation reactions. To avoid problems caused by residual
detergent in glassware, use disposable plastic tubes and flasks wherever possible for preparation and storage of all solutions
and media used in transformation. Organic contaminants present in the H2O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H2O obtained directly from a well-serviced Milli-Q filtration system (Millipore) usually gives good results. If problems should
arise, treat the deionized H2O with activated charcoal before use.
|
| If necessary, adjust the pH of the final solution to 6.4 with 0.1 m HCl. This prevents precipitation of MnO2. Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-µm pore size). Divide the solution
into 40-mL aliquots in tissue-culture flasks (e.g., Corning or equivalent) and store them at 4°C. During storage, the pH of
the solution drifts down to a final value of 6.1–6.2 but then stabilizes. During prolonged storage, a faint precipitate of
a tan-colored substance may appear. It looks ugly but does not seem to affect the efficiency of transformation.
|
