ARNT Knockout genotyping by PCR
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Bradfield Lab, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School
http://mcardle.oncology.wisc.edu/bradfield/ARNT-Knockout.html
Amplify a 459bp fragment of neomycin using OL855 and OL856. The neomycin gene will only be present in the knockout allele. Progeny will be either +/+, or +/- ( -/- do not survive), so a positive PCR reaction indicates ARNT +/- and a negative PCR indicates ARNT +/+. Run 15 ul from each reaction on a 1% agarose gel.
Master Mix:
5ul 10x PCR buffer ( with MgCl2 1.5mM final)
4 ul dNTP mix (2.5 mM each)
1 ul OL855 (5 uM)
1 ul OL856 (5 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 -> 62 C/1min -> 72 C/1min]x30 -> 72 C/5min
Primer sequences
OL855
5’-ggc gcg agc ccc tga tgc tc-3’
OL856
5’-ttg ggt gga gag gct att cgg cta tga c-3’
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