Tempel Lab Protocols

PATCH GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
PTC F
PTC R

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

PTC F GAT GTT TCG CTT GGT GGT CGA ATG
PTC R GCT GTT CTC CTC TTC CTC ATC TCC

Procedure

The primers recognize the PATCH mutant and not the Wildtype.

PCR Rxn
10x PCR buffer 2.5 ul
10mM Nucleotide mix 0.5 ul
PTC F primer 20 uM 1 ul
PTC R primer 20 uM 1 ul
Sterile Water 18.8 ul
FastStart TAQ 0.2 ul
Tail Lysate 1 ul
Total Volume 25 ul
PCR Protocol

94 deg C
4:00
94 deg C
 0:30
64 deg C
0:30
x35
72 deg C
2:00
72 deg C 7:00
4 deg C Hold

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

Bands
Wildtype: No Band
Mutant: 400-500 bp