PCR Typing Ear Punches

 

Heat ear punch in 300 µl 10 mM NaOH/0.1 mM EDTA at 95°C for 10 min. Store at RT.

(300 µl 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 µl of each DNA sample with 9 µl of water in each PCR reaction. Denature at 95°C for 10 minutes in the PCR machine. At 85°C add 7 µl of the cocktail (freshly made and premixed; can be added through the oil.)

Cocktail per reaction

	100 ng/µl primer 1	1.2 µl
	100 ng/µl primer 2	1.2 µl
	10X PCR buffer	2.0 µl
	20 mM MgCl2	2.0 µl
	10 mM dNTPs	0.4 µl
	5 U/µl Taq	0.2 µl

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.