Tempel Lab Protocols

DFW3J GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
dfw3j Wild Type Forward
dfw3j Mutant Forwad
dfw3j Common Reverse

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

dfw3J wtF CCA TCC TGC TGT CAG TCA TC
dfw3J mtF GCC ATC CTG CTG TCA GTT G
dfw3J comR TGG TGA AGC TGT GTT GGA AG

Procedure

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

PCR Rxn
Sterile Water 17.8 ul
10x PCR buffer w/ MgCl2 2.5ul
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
60 deg C
0:30
x30
72 deg C
0:45
72 deg C 7:00
4 deg C Hold

Gel
1.5 % Agarose + 2 ul EtBr (10mg/ml)

Bands
wildtype: 500bp
mutant: 500bp