Tempel Lab Protocols

QV 4J GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
18F QV-4J Forward
18R QV-4J Reverse

PCR Reagents:
Sterile water
10x buffer w/MgCl2
10mM Nucleotide mix
FastStart Taq

Restriction Digest Reagents
Sfi 1

Primer Sequences

Qv4JF TGT CTC TCC TGT TGT CAC GG
Qv4JR AGC CTC AGG TTG AAA CAC CC

Procedure

Run PCR on 2ul of lysed tail DNA, diluted 1:4. Digest with Sfi 1, which recognizes and cuts a site in the wildtype, but not the mutant.

PCR Rxn
Sterile Water 37.6 ul
10x PCR buffer w/MgCl2 5 ul
10mM Nucleotide mix 1 ul
18 F primer 20 uM 2 ul
18 R primer 20 uM 2 ul
FastStart Taq 0.4 ul
Tail Lysate (diluted 1:4) 2 ul
Total Volume 50 ul
PCR Protocol

95 deg C 4:30
95 deg C
 0:40
63 deg C
0:30
x35
72 deg C
0:35
72 deg C 7:00
4 deg C Hold

Sfi 1 Digest
Sterile Water 2.05 ul
BSA (100x) 0.25 ul
10x Buffer
2.5 ul
Sfi 1 Enzyme 0.2 ul
PCR Product 20 ul
Total Volume 25 ul

Digest for 3 hours @ 50 deg. C

Gel
1.5 % Agarose + 3 ul EtBr (10mg/ml)

Bands
Uncut PCR Product: 430
Wildtype: <430 bp
Mutant: 430 bp,

U = Uncut DNA, S = Sfi 1 digests