Tempel Lab Protocols

Prestin CRE GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
Cre Forward
Cre Reverse

PCR Reagents:
Sterile water
10x buffer w/MgCl2
10mM Nucleotide mix
FastStart taq
Primer Sequences

Cre F AAC ATT CTC CCA CCG TCA GT
Cre R CAT TTG GGC CAG CTA AAC AT

Procedure
The primers recognize the mutant and not the wildtype.

PCR Rxn
Sterile Water

17.8 ul
10x PCR buffer w/MgCl2 2.5ul
10mM Nucleotide mix 0.5 ul
Cre Forward primer (20 uM) 1 ul
Cre Reverse primer (20 uM) 1 ul
FastStart Taq 0.2ul
Tail Lysate 2 ul
Total Volume 25 ul
PCR Protocol

95 deg C 4:30
95 deg C
 0:30
56 deg C
 0:45 x30
72 deg C
 0:45
72 deg C 7:00
4 deg C Hold

Gel
2% Agarose + 3 ul EtBr (10mg/ml)

Bands
WildType: No band
Mutant: 400 bp