Material
- proteinase K
- Taq 10x buffer
- tabletop shaker/incubator
Procedure
tissue lysis to release DNA
per tail or tissue chunk add (tissue degradation is sped up if tissue/tail is cut into smaller bits):
- 100 µl Taq DNA polymerase 10x Reaction buffer with MgCl2 (e.g. Promega #M1883) diluted 1:10 in ddH2O
- +2 µl proteinase K (20 mg/ml)
- incubate for 3h to o/n at 50°C at 300 rpm
- (proteinase K is stable over a broad pH range (4.0 to 12.5, optimum pH 8.0) and is also stable over the temperature range of 25 to 65°C)
proteinase K inactivation
- 95°C for 10-20 min at 300 rpm
- centrifuge at 13000 rpm for 3 min and transfer supernatant (80 µl) into new eppi
storage before PCR
- 4°C short term, -20°C long term