Tempel Lab Protocols

DFW2J GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
dfw2j Wildtype Forward
dfw2j Mutant Forward
dfw2j Common Reverse

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

dfw 2j wtF CCA TCT GCT CAG ACA AGA CAG
dfw 2j mtF CCA TCT GCT CAG ACA AGA CGA
dfw 2j comR GAC ACT GCC CCG TGT CTA CT

Procedure

Run the wildtype (wtF& comR primers) and mutant (mtF & comR primers) PCR reactions in separate tubes.

PCR Rxn
Sterile Water 17.8 ul
10x PCR buffer w/MgCl2 2.5 ul
10mM Nucleotide mix 0.5 ul
Forward primer 20 uM: wtF or mtF 1 ul
Common Reverse primer 20 uM 1 ul
FastStart Taq 0.2 ul
Tail Lysate 2 ul
Total Volume 25ul
PCR Protocol

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

Gel
1 % Agarose + 2 ul EtBr (10mg/ml)

Bands
Mutant: 500 bp
Wildtype: 500bp