Protocol 21285: Probe Assay - Greb1l<em1(IMPC)J> Probe Alt 1
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

Mut= 99 bp

Wt= 121 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31220 TGG GAT GCT CCA AAG AAA GG Common A
31222 CAA TAA TGA CTA AAA ATG AAA ACC AAA Mutant Reverse A
31223 Fluorophore-1 CGT TTC CTC CTC TTA ACT CAG C Quencher-1 WT Probe
31224 Fluorophore-2 TTC CTT GCC CCT GAA ATT CT Quencher-2 MUT Probe
32130 TGT TGA CCA ATA CTG GGC ACT AAC Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31220 0.40 uM
31222 0.40 uM
32130 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.