Ahr 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/Ahr-knockout.html
This protocol is published in Journal of Biological Chemistry (1993) 268:22203-22209.
PCR a 218bp fragment of exon 7 using OL72 and OL111. Both of the following protocols work equally as well so pick the one that sounds the best to you. After you have your PCR product you will need to digest the fragment with Eco47III, HaeII, or HhaI. (FYI-HaeII and HhaI are much less expensive than Eco47III.) The “d” allele does not cut with the enzyme due to a polymorphism at that site, but the b1 allele cuts once to yield 142bp and 76bp fragments. The Ahr was generated in ES cells from a 129 mouse strain which has the “d” allele. The BL6 mouse which we have bred onto has the “b” allele. Progeny will be +/+=b/b,
+/-=b/d, or -/-=d/d.
Ahr KO using Amplitaq Gold
Master Mix:
10 ul Perkin-Elmer 10x buffer with MgC12
2 ul of each dNTP
2 ul Ol 72 (5 uM) This is the concentration of the oligo
2 ul Ol 111 (5uM) " " " " " " "
75 ul distilled, deionized, sterile water
1 ul Ampiltaq Gold
UV irradiate pipettes, pipette tips, gloves, tube for master mix, water, and PCR tubes
Make the master mix on ice and aliquot 99 ul to tubes on ice
Add 100 ng of template to the PCR tubes and add to a pre-heated block
This should be a 100 ul reaction
Cycle parameters:
95 C/10min -> [95 C/1min -> 55 C/1min -> 72 C/10sec]x 35 -> 72 C/15min
Ahr KO using Promega Taq
Master Mix:
5 ul Promega 10 x buffer without MgCl2
4 ul 25 mMgC12
1 ul of each dNTP
1.5 ul Ol 72 (5 uM)
1.5 ul Ol 111 (5 uM)
29.5 ul distilled, deionized, sterile water
UV irradiate pipettes, pipette tips, gloves, tube for master mix, water, and PCR tubes Make the master mix on ice and aliquot 45.5ul to tubes on ice. Add 400 ng of template to the PCR tubes and add to a pre-heated block. Let denature for about 8 minutes and then add 0.5 ul of Promega taq to the reaction tubes.
This should be a 50 ul reaction
Cycle parameters: 95 C/10min -> [95 C/1min -> 63 C/1min -> 72 C/20sec]x 35 -> 72 C/5min
These PCR protocols have been optimized on the MJ PCR machine.
Primer sequences
OL 72
5’-ggt tcg aat ttc cag gat gg-3’
OL111
5’-cca ccc cag gta cat gat gga acc-3’
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