Tempel Lab Protocols

Kcna2 (Kv1.2) GENOTYPE

You will Need

Mouse Tail Digests

4 Primers:
Kv1.2 Wildtype Forward
Kv1.2 Wildtype Reverse
Neo1 Mutant Forward
Neo6 Mutant Reverse

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

KV1.2 F CGT CCT CCC CTG ACC TAA AG
KV1.2 R CAT TAG TAG GCT TCA ATC AGA CAT CA
Neo1 ATC TCC TGT CAT CTC ACC TTG C
Neo6 GCC AAC GCT ATG TCC TGA TAG C

Procedure

Run the wildtype (kv1.2 F & kv1.2 R primers) and the mutant (neo1 & neo6 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 kv1.2F wt or neo1mt 1ul
Reverse primer 20 uM kv1.2 R wt or neo6 mt 1ul
FastStart Taq 0.2ul
Tail Lysate 2ul
Total Volume 25ul
PCR Protocol

94 deg C 4:30
94 deg C
 0:30
60 deg C 0:45 x30
72 deg C 0:45
72 deg C 7:00
4 deg C Hold

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

Bands
WildType 210 bp
Mutant 470 bp