PCR Typing Ear Punches
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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.
![PSMB9 LMP2=proteasome LMP2.s {alternatively spliced} [human, EBV-transformed B lymphoblastoid typing cell lines, mRNA, 645 nt].](https://img1.dxycdn.com/p/s14/2024/0514/752/2760093895118520971.jpg!wh200)
