Tempel Lab Protocols

QVnot GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
28F Forward
28R Reverse

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

Restriction Digest Reagents
Nmuc1

Primer Sequences

28F GTC TCC ATT ACT CCT AAG GC
28R ATT CTG CCC AAA GCT CTG GG

Procedure

Run a touchdown PCR program that decreases the primer annealing temperature by 0.3 deg. C per cycle for 25 cycles with an additional 15 cycles at 58 deg. C
Digest 20 ul of the PCR Product with Nmuc1 at 37 deg. C for 3 hours.

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

94 deg C 4:30
94 deg C
 0:30
66 deg C
0:30
x25
drop by 0.3 degree C per cycle
72 deg C
1:00
94 deg C
 0:30
58 deg C
 0:30

x15
72 deg C
 1:00
72 deg C 7:00
4 deg C Hold

Nmuc1 Digest
10x Nmuc1 Buffer 2.25 ul
Nmuc1 Enzyme 0.2 ul
PCR Product
20 ul
Total Volume 22.45 ul

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

Bands
Wildtype: 415 bp
Mutant: 246 bp, 169 bp

U= uncut PCR product, N= Nmucl digest