Protocol 21428: Probe Assay - Chmp7<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=  116 bp

Wild Type = 117 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32426 GGG TAA GAA GAG CCC AGG A Common A
32427 Fluorophore-1 AGG CTG TCT GTC GCA TTA CA Quencher-1 WT Probe
32428 Fluorophore-2 CAT TTC CAG GGT GAG CTT TG Quencher-2 MUT Probe
32429 CCA CAC TGG CCA TGA AGT C Wild type Reverse A
32430 CAC TGC AGC TAA TCA TTA ATG TCC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32426 0.40 uM
32429 0.40 uM
32430 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.