Amplify a 700bp fragment using OL637 and OL639. Run 10 ul from each reaction on a 1% agarose gel.
Master Mix:
5 ul 10xPCR buffer (with MgCl2, 1.5mM final)
4 ul dNTP mix (2.5 mM each)
1 ul OL637 (20 uM)
1 ul OL639 (20 uM)
0.5 ul Taq
34.5 ul distilled, deionized, sterile water
4 ul mouse tail DNA
Typically you will be genotyping several animals at once, so make a master mix of everything but the DNA. Aliquot 46 ul to tube and add 4 ul of DNA. Set the reactions up on ice, and immediately put them into the thermalcyler which has been preheated to 95 C. Remember to run a water control. This should be a 50 ul reaction. If you are having problems with contamination try a hot start.
Cycle parameters:
95 C/5min -> [95 C/1min -> 57 C/1min -> 72 C/1min]x32 -> 72 C/5min
Primer sequences
OL637
5’-gaa gat ctt cat gct gtc cat gct ggg aga t-3’
OL639
5’-gaa gat ctt caa ggc act gcc ctc ttg aag c-3’
