Tempel Lab Protocols

QV 3J GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
Qv3JF Forward
Qv3JR Reverse

PCR Reagents:
10x buffer
10mM Nucleotide mix
Sterile water
Fast Taq

Restriction Digest Reagents
Sty 1

Primer Sequences

Qv3JF GAA ACT CAA GGC AGA ACA GAG TAA
Qv3JR TCG CTA GAC TCC TGT CGT TCT AT

Procedure

Run PCR on a 1:5 dilution of digested tail DNA. Digest with Sty 1 which recognizes one site in the Wildtype and 2 sites in the Mutant.

PCR Rxn
10x PCR buffer 5 ul
10mM Nucleotide mix 1 ul
28 F primer 20 uM 2 ul
28 R primer 20 uM 2 ul
Sterile Water 38.5 ul
FastStart TAQ 0.5 ul
Tail Lysate (diluted 1:5) 3 ul
Total Volume 50 ul
PCR Protocol

95 deg C 4:30
95 deg C
 0:40
63 deg C
0:30
x25
72 deg C
0:35
72 deg C 7:00
4 deg C Hold

Sty1 Digest
10x Buffer 5 ul
Nmuc1 Enzyme 0.5 ul
Sterile Water
14 ul
PCR Product 30ul
BSA (100x) 0.5 ul
Total Volume 50 ul

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

Bands
Uncut PCR Product: 579
Wildtype: 83 & 496 bp
Mutant: 83, 229 & 266 bp,

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