| MnCl2·4H2O
|
10.88 g |
55 mm |
| CaCl2·2H2O
|
2.20 g |
15 mm |
| KCl |
18.65 g |
250 mm |
| PIPES (0.5 M, pH 6.7) |
20 mL |
10 mm |
| H2O
|
800 mL |
|
| Dissolve the solutes in 800 mL of pure H2O. Then add 20 mL of 0.5 m PIPES (pH 6.7). Adjust the volume of the buffer to 1 L with pure H2O. Sterilize by filtration through a prerinsed 0.45 µm Nalgene filter. Divide into aliquots and store at −20°C.Prepare 0.5
m PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 mL of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 m KOH, and then add pure H2O to bring the final volume to 100 mL. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter
(0.45-µm pore size). Divide into aliquots and store frozen at −20°C.Organic contaminants 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.
|
