Protocol 31361: Probe Assay - Gabrr3<em1(IMPC)J> 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=  105 bp

Wild Type = 92 bp

>chr16:59429784+59429875 92bp TGAGAGACCAGCTCCATCCT GCATTATGAACTCGCTGACG

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36702 TGA GAG ACC AGC TCC ATC CT Common A
36703 TCT ACA GAT TTC CAG AGG TCA GC Mutant Reverse A
36704 GCA TTA TGA ACT CGC TGA CG Wild type Reverse A
36705 Fluorophore-1 TGT TCT GTA TAT TCC TGC TCT GTC C Quencher-1 MUT Probe
36706 Fluorophore-2 ATG ATA TGC AAG CCA TGA AGA GTA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36702 0.40 uM
36703 0.40 uM
36704 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.