Heat ear punch in 300 �l 10 mM NaOH/0.1 mM EDTA at 95°C for 10 min. Store at RT.
(300 �l aliquots of 10 mM NaOH/0.1 mM EDTA can be frozen. However, do not store 10 mM NaOH/0.1 mM EDTA at room temperature in glass, as it will react with the glass.)
Use 4 �l of each DNA sample with 9 �l of water in each PCR reaction. Denature at 95°C for 10 minutes in the PCR machine. At 85°C add 7 �l of the cocktail (freshly made and premixed; can be added through the oil.)
Cocktail per reaction
| 100 ng/�l primer 1 | 1.2 �l | |
| 100 ng/�l primer 2 | 1.2 �l | |
| 10X PCR buffer | 2.0 �l | |
| 20 mM MgCl2 | 2.0 �l | |
| 10 mM dNTPs | 0.4 �l | |
| 5 U/�l Taq | 0.2 �l |
Cycle conditions
| 94°C |
0.5 min |
|
| 62°C |
0.5 min |
|
| 72°C |
1.5 min |
|
| 39 cycles |
Run the whole sample on an agarose gel with markers, a no DNA negative control, and positive controls. The primers are in massive excess and will make a low molecular weight smear on the gel.