Protocol 21269: Probe Assay - Brip1<em1J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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= 133 bp

Wild Type = 143 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32103 TTG ACA TGT TTA TAA AAC CAG CAG A Mutant Forward A
32104 Fluorophore-1 TGT TCA GAA GAA ATG TAA AGG AAT AAA Quencher-1 MUT Probe
32105 ATG TGC TGT GGG GTG GAG Wild type Forward A
32106 AAC TAG CAG TCT GAA ATA TCA AGA AGT Common A
32107 Fluorophore-2 CAC TCC TCA CCT TCC ACC ATA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32103 0.40 uM
32105 0.40 uM
32106 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.