Tempel Lab Protocols

Kcna1 (Kv1.1) GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
905 Wildtype Forward
906 Common Reverse
907 Mutant Forward

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

905 TAG CCT CTG ACA GTG ACC TCA GC
906 GAA AGC TTC AGG TTC GCC ACT CC
907 CGC CTT CTA TCG CCT TCT TGA CG

Procedure
Run the wildtype (905 & 906 primers) and the mutant (906 & 907 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
Forward primer 20 uM 905WT and 907MT 1 ul
Common reverse primer (906) 20 uM 1ul
FastStart Taq 0.2ul
Tail Lysate 2 ul
Total Volume 25ul
PCR Protocol

95 deg C 4:30
95 deg C 0:30
63 deg C 0:30 x30
72 deg C 0:35
72 deg C 7:00
4 deg C Hold

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

Bands
WildType 344 bp
Mutant 504 bp