- Dissect yolk sac and rinse thoroughly in PBS.
- Place yolk sac or ear punch into 100 to 300 µl ProtK buffer with non-ionic detergents.
- Incubate overnight at 55°C.
- Place 5 µl of the ProtK’d material into a PCR tube with 30 µl dH20, overlay with two drops of mineral oil. Too much tissue can inhibit the PCR.
- Run PCR reaction at 95°C for ten minutes to inactivate ProtK.
- Bring the PCR machine to soak at your annealing temperature and add PCR mix 15 ul.
- Begin PCR cycles.
ProtK Buffer with Non-ionic Detergents
| Final Concentration | For 100 ml |
| 50 mM KCl | 5 ml of 1 M stock |
| 10 mM Tris.HCl (pH 8.3) | 1 ml of 1 M stock |
| 2.0 mM MgCl2 | 0.2 ml of 1 M stock |
| 0.1 mg/ml gelatin | 10 ml 1% stock |
| 0.45% Nonidet P-40 | 0.45 ml |
| 0.45% Tween-20 l | 0.45 ml |
| H2O | 83 ml |
Autoclave solution, aliquot and store frozen. Thaw immediately before use and add 25 ul ProK 20mg/ml stock solution per 1 ml of buffer.
| PCR Mix - Stocks | One Reaction | Ten Reactions (Make for 11 Reactions) |
| dNTP 2 mM | 5 µl | 55 µl |
| 10x PCR Buffer | 4.5 µl | 49.5 µl |
| Primers 25 uM | 1 µl each | 11 µl each |
| Taq Polymerase | 0.25 µl | 2.75 µl |
| H2O | 3.25 µl | 35.75 µl |
| Total Volume | 15 µl | 165 µl |