Protocol 32429 - Kcnq2<em3(T274M)Frk>

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mut=114 bp

Wt=114 bp

Sequence

Wt Sequence (bp changes in brackets with wt first):

ATTGCGACCTCTGTGGTAGGCCATTTCCAGCCTGCCCAAGTAACCAGAGCCCCTTACCCCTCAGATCA(cc/tg)CTGACGACCATTGGCTACGGGGACAAGTACCCTCAGACCTGGAA

Kcnq2 T274M (ACC -> ATG)

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
39097		ATT GCG ACC TCT GTG GTA GG		Forward	A
39098		TTC CAG GTC TGA GGG TAC TTG		Reverse	A
39099	Fluorophore-1	TAC CCC TCA GAT CAC CCT GA	Quencher-1	WT Probe
39100	Fluorophore-2	CCT TAC CCC TCA GAT CAT GC	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
39097	0.40 uM
39098	0.40 uM
Wt Probe	0.15 uM
Mutant Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--
4		--	repeat steps 2-3 for 40 cycles
5	40.0	--	Forever

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.