Heat ear punch in 300 Ál 10 mM NaOH/0.1 mM EDTA at 95°C for 10 min. Store at RT.
(300 Ál 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 Ál of each DNA sample with 9 Ál of water in each PCR reaction. Denature at 95°C for 10 minutes in the PCR machine. At 85°C add 7 Ál of the cocktail (freshly made and premixed; can be added through the oil.)
Cocktail per reaction
|100 ng/Ál primer 1||1.2 Ál|
|100 ng/Ál primer 2||1.2 Ál|
|10X PCR buffer||2.0 Ál|
|20 mM MgCl2||2.0 Ál|
|10 mM dNTPs||0.4 Ál|
|5 U/Ál Taq||0.2 Ál|
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.