Bradfield Lab, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School
http://mcardle.oncology.wisc.edu/bradfield/Ahr-transgenic-genotyping.html
Amplify a 600bp fragment using OL638 and OL639. Run 10 ul from each PCR reaction out on a 1% agarose gel.
Master Mix:
5 ul 10xPCR buffer (with MgCl2, 1.5 mM final)
4 ul dNTP mix (2.5 mM each)
1 ul OL638 (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.
Cycling parameters:
95 -> C/5min -> [95 C/1min -> 60 C/1min -> 72 C/1min]x32 -> 72 C/5min
Primer sequence
OL638
5’-gaa gat ctt cat ctt cag cca ctt cat cat cc-3’
OL639
See above sequence
