Stock No: 020653
Protocol 21930: End Point Analysis Assay - Cacna1a<rkr>-EP
Version 3.0

Notes

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 = A/A
Heterozygote = A/C
Wild type = C/C

Sequence

CCATACCAATGTTTTCAGCTCACCCaaccccaccaccacgccctccccgcctcacacccctcctcctgccgcaggccgtgtttgactgcgtggtgaactcgctcaagaacgtcttcaa

catcctcattgtgtacatgctgttcatgttcatcttcgctgtggtagccgtgcagctcttcaagggcaagttcttccactgca[C/A]ggacgagtccacggagttcgagagagactgtcggtgggtggga

cacgcggcaggggaggcggggtccagggcccacagtcacacactgaggcagagcggtaactccgggtcagcttggatttttagagtcaggtggggtatacctaggaacctcagcactcaga

aggctgaggcaggaggatcacCCTGAGTTTTGAGACCAAGCTGGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
17030 CAT GTT CAT CTT CGC TGT GG Forward A
17031 GCT CTG CCT CAG TGT GTG AC Reverse A
17032 Fluorophore-1 CCA CTG CAC GGA CGA GT Quencher-1 WT Probe
17033 Fluorophore-2 TCC ACT GCA AGG ACG AGT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
17030 0.40 uM
17031 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.