ARNT transgenic genotyping by PCR

Bradfield Lab, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School
http://mcardle.oncology.wisc.edu/bradfield/ARNT-transgenic-genotyping.html
 
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’

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