Stock No: 030234
Protocol 22032: Probe Assay - C7<em1J> Probe-Alternate 2
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=131  bp

Wt= 142 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32198 Fluorophore-1 TCT CCT AGG TGA CAA GCT TAT TAA TTT Quencher-1 WT Probe
32200 CGC TGC TAT AAA GCA CAC CA Wild type Reverse A
32978 Fluorophore-2 AGA AAC AAG GAC AAA AAT TCC TCA Quencher-2 MUT Probe
32979 TGG CTC TTC CTC TCA TCT CC Common A
33556 TGC AGC TCT CTG AAT GAA AGT Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32200 0.40 uM
32979 0.40 uM
33556 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.