APC flox PCR

APC-flox (Exon 1-14) 5′

Reaction Mix:

Tail DNA1.0 ul
10x PCR buffer2.0 ul
10mM dNTPs0.4 ul
Flox 1 (20uM)0.2 ul
Flox 2 (20uM)0.2 ul
milliQ15.2 ul
House Taq1.0 ul

PCR Primers:

Flox 15′ GAT CAC TCA TCC GAT AAG TGC 3′
Flox 25’ TTG GTT AAG GTG GTC TTG CAG 3’

PCR Conditions:

1.94C3:00
2.94C0:30
3.56C1:00
4.72C0:30
5.Goto 2 x30 cycles
6.72C10:00
7.4C0:00
END

Expected Bands:

Wt = 340 bp
1lox at 5′-end of Apc = 471 bp

APC-flox (Exon 1-14) 3′

Reaction Mix:

Tail DNA1.0 ul
10x PCR buffer2.0 ul
10mM dNTPs0.4 ul
Flox 3 (20uM)0.2 ul
Flox 4 (20uM)0.2 ul
milliQ15.2 ul
House Taq1.0 ul

PCR Primers:

Flox 35′ TGA CAG CAC AGA ATC CAG TG 3′
Flox 45’TAC CAA GCA TTG AGA G 3’

PCR Conditions:

1.94C3:00
2.94C0:30
3.56C1:00
4.72C0:30
5.Goto 2 x30 cycles
6.72C10:00
7.4C0:00
END

Expected Bands:

Wt = 1.1 Kb
1lox1 frt at 3′-end = 1.2 Kb

APC-null

Reaction Mix:

Tail DNA2.0 ul
10x PCR buffer2.0 ul
10mM dNTPs0.4 ul
Flox 1 (20uM)0.2 ul
Flox 4 (20uM)0.2 ul
milliQ14.2 ul
House Taq1.0 ul

PCR Primers:

Flox 15′ GAT CAC TCA TCC GAT AAG TGC 3′
Flox 45’TAC CAA GCA TTG AGA G 3’

PCR Conditions:

1.94C3:00
2.94C0:30
3.55C1:00
4.72C1:00
5.Goto 2 x35 cycles
6.72C5:00
7.4C0:00
END

Expected Bands:

null = 1.25 Kb
WT or still floxed = no band

Notes:

It is possible to get Apc-fle1-14 homozygotes and breed them, but the problem is that one can’t distinguish heterozygous from homozygous conditional mutants by PCR genotyping. The WT band is always there in every genotyping protocol we’ve tried, or some contaminating band that is the same size as the wt band. We are not sure why. We tried lots of primer pairs and we do know that the targeting is correct, though, so no worries about that being the reason.

The way we breed homozygous mutants is by crossing to Apc-Min or other mutant Apc alleles. For example, progeny of Apc-fle1-14/Min x Apc-fle1-14/Min that didn’t carry Min (Min genotyping protocol yields “WT”) were Apc-fle1-14/fle1-14 homozygous.

Sorry that this sounds like a complete pain, and we seriously regret that we never got a genotyping protocol that worked. Find below files for sequence information for WT and Apc-fle1-14 allele in case you’d like to try to devise a genotyping protocol of your own. If you manage to find something that works, we’d love to hear about it. Good luck!

10X PCR Buffer:

500 mM KCl
100mM Tris pH 8.3
15 mM MgCl2
1 mg/ml BSA
2 mM dNTPs
dH20

(store at -20ºC)

Scroll to Top