PCR Genotyping Ear Punches

PCR Typing Ear Punches

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

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.