Protocols
Ear punches/yolk sacs/toes DNA for PCR
- Dissect yolk sac and rinse thoroughly in PBS.
- Place yolk sac or ear punch into 100-300 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 10 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
50 mM KCl
10 mM Tris.HCl (pH 8.3)
2.0 mM MgCl2
0.1 mg/ml gelatin
0.45% Nonidet P-40
0.45% Tween-20 l
H20
5 ml of 1 M stock
1 ml of 1 M stock
0.2 ml of 1 M stock
10 ml 1% stock
0.45 ml
0.45 ml
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
dNTP 2 mM
10x PCR buffer
Primers 25 uM
Taq polymerase
H2O
Total volume
one reaction
5 µl
4.5 µl
1 µl each
0.25 µl
3.25 µl
15 µl
(make for 11 reactions)
55 µl
49.5 µl
11 µl each
2.75 µl
35.75 µl
165 µl