Tempel Lab Protocols

DFW GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
dfw Common Forward
dfw Wild type Reverse
dfw Mutant Reverse

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

dfw comF CAG ACC CCT GAA TTC TCC AT
dfw wtR AAG CAG GGT AAA TAT GAT GCC
dfw mtR CAA GCA GGG TAA ATA TGA TGC T

Procedure

Run the wildtype (comF & wtR primers) and the mutant (comF & mtR primers) PCR reactions in seperate tubes.

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

PCR Protocol

94 deg C 4:30
94 deg C
 0:30
64 deg C
0:30
x36
72 deg C
1:00
72 deg C 7:00
4 deg C Hold

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

Bands
Wildtype: 500 bp
Mutant: 500bp