Protocol 21304: Probe Assay - Gpd1l<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=  103 bp

Wild Type = 114 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32165 CGC TCA CTT GAG TGG ACT TG Common A
32166 Fluorophore-1 TGC ATT TTT GCC TTT GAG TG Quencher-1 WT Probe
32167 Fluorophore-2 TCT TGG ATT TGC GTT GGT TT Quencher-2 MUT Probe
32168 AAC AGC TGA TCC CCT GGA A Wild type Reverse A
32169 GGA CCA ATA GTA TCC ACA GAA GAA A Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32165 0.40 uM
32168 0.40 uM
32169 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.