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Ear Punches/Yolk Sacs/Tail DNA for PCR

  • Dissect yolk sac and rinse thoroughly in PBS.
  • Place yolk sac or ear punch into 100 to 300 l ProtK buffer with non-ionic detergents.
  • Incubate overnight at 55C.
  • Place 5 l of the ProtK’d material into a PCR tube with 30 l dH20, overlay with two drops of mineral oil. Too much tissue can inhibit the PCR.
  • Run PCR reaction at 95C 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 l 55 l
10x PCR Buffer 4.5 l 49.5 l
Primers 25 uM 1 l each 11 l each
Taq Polymerase 0.25 l 2.75 l
H2O 3.25 l 35.75 l
Total Volume 15 l 165 l