This is a cached page for the URL (http://transgenic.cwru.edu/protocols/pcrtypingearpunches.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
PCR Typing 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.