Protocol 46083: Probe Assay - Cacna1a<em#Lutzy>
Version 1.0

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

Mutant=  110 bp

Wild Type = 110 bp

Sequence

Wt Sequence:

GTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGTCAATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT

Mutant Sequence:

GCTCCATGGGTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGctgATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
67170 GGG TGC TTG CTT ACA GAC C Forward A
67172 AAC AGG AGG AGG CCG ATC T Reverse A
67173 Fluorophore-1 CGT CCT GAA GTC AAT CAT GAA G Quencher-1 WT Probe
67175 Fluorophore-2 CGT CCT GAA GCT GAT CAT GAA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
67170 0.50 uM
67172 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.