This method is modified from Dr. Lyn Corcoran (WEHI)
DNA buffer : 1 X PCR buffer
0.45% NP - 40
0.45% Tween - 20
Proteinase K 100 mg / ml
Prepare the DNA for PCR :
1. In PCR tube contain 50ul DNA buffer,
2. Dig washed york sack into tube, spin down,
3. Incubate at 500C for 30 minutes,
4. Boil the lysate for 5 minutes, spin down,
5. Take 5ul for PCR.
PCR reaction (50ul) contains :
1 X PCR buffer
2 mM MgCl2
100 nM dNTPs
Primers : a. 0.25uM
b. 0.05uM
c. 0.2uM
a c 400bp (targeting region)
a b 200bp (wild type)
2.5 unite/ 100ulTaq DNA polymerase
PCR cycle : Initial denaturing by 940C for 4 minutes
28 cycles of 940C for 1 minute
600C for 1 minute
720C for 1 minute
Final extension by 720C for 10 minutes