| Creatine phosphate (0.5 m)
|
10 µL |
20 mm |
| ATP (100 mm)
|
5 µL |
2 mm |
| KCl (3 m)
|
2.5 µL |
30 mm*
|
| MgCl2 (0.5 m)
|
2.1 µL |
4.2 mm |
| Dithiothreitol (DTT) (1 m)
|
1 µL |
4 mm |
| Tris (1 M, pH 8.0) |
1 µL |
16 mm |
| Aprotinin and leupeptin stock solution** |
1 µL |
|
| Polyethyleneglycol (PEG 8000; 20%, w/v)*** |
37.5 µL |
3% |
| H2O
|
2.1 µL |
|
|
|
|
| Prepare fresh and keep on ice. |
| *The final KCl concentration in the reaction mixture is usually between 50 and 80 mm because the splicing extract also contains KCl, which contributes to the final concentration. Adjust according to the concentration
in dialysis buffer and the amount of extract used in the reaction.
|
| **Prepare the aprotinin and leupeptin stock solution immediately before use by adding 1 µL of a 10-mg/mL solution of aprotinin
and 1 µL of a 2-mg/mL solution of leupeptin to 38 µL of H2O.
|
| ***Weigh out 8 g of PEG 8000 (e.g., Carbowax PEG 8000 from Fisher) in a 50-mL conical tube. Add RNase-free H2O to the 40-mL mark on the tube. Close the lid tightly and heat briefly at 65°C to dissolve. Store for 4 mo at room temperature.
|
