This protocol uses Ampitaq Gold from Perkin-Elmer, which eliminates the need for a manual hot start. The protocol will work just as well using the taq of your choice and a manual hot start. If you are not going to use Amplitaq Gold you will need to adjust the annealing temperature (higher). The protocol uses three oligos that will distinguish between +/+, +/-, and -/-. The 0.9kb band is the -/-, the 1.2kb band is the +/+, and both the 0.9kb and 1.2kb bands are the +/-. To get the best results read your sample on a spectrophotometer and then dilute your sample to 100ng/ul.
5ul PCR reaction buffer with MgCl2 (15mM)
0.75ul Ol 1506 (25uM)
0.75ul Ol 1507 (25uM)
0.75ul Ol 1508 (25uM)
0.5 ul Amplitaq Gold
1.0 ul of each dNTP
34.25 ul of distilled, deionized, sterile water
aliquot 46 ul
4 ul DNA template (~400ng)
50 ul reaction
Cycle parameters:
94 C/10mi -> [94 C/1min -> 55 C/2min -> 72 C/3min]x33 -> 72 C/5min
Primer sequences
OL1506
5’-cag cct ggg atg gaa atc aag aca-3’
OL1507
5’-cgc tgc aca cgg cac tct gag tac-3’
OL1508
5’-agc gcc tcc cct acc cgg tag aat-3’
