Tempel Lab Protocols

DFW4J GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
E6 CBA Wildtype Forward
E6 B6 Mutant Forward
E6 Common Reverse

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

E6 CBA wtF CCC ATG TTG CTT TCA GGT AAG TG
E6 B6 mtF CCA TGT TGC TGT CAG GTA AGC T
E6 comR CTT CCC ATT GCC TCC TCT GT

Procedure

Run the wildtype (E6 CBAF & E6 comR primers) and the mutant (E6 B6 & E6 comR primers) PCR reactions in separate tubes.

PCR Rxn
Sterile Water 17.8ul
10x PCR buffer w/ MgCl2 2.5 ul
10mM Nucleotide mix 0.5 ul
Forward primer 20 uM 1 ul
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
x36
72 deg C
0:45
72 deg C 7:00
4 deg C Hold

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

Bands
Mutant: 500bp
Wildtype: 500bp