Stock No: 002984
Protocol 21774: Probe Assay - Il12rb1<tm1Jm> 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=  92 bp

Wild Type = 101 bp

Sequence

Wt Sequence: ctatgtagcccaggctgtcctcaaagtcacactcacagagatcctcctgcctctgactccCGaattctgggattaaacagcggttgggattagactgcgtc

 

Mutant Sequence: CTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAgaattctgggattaaacagcggttgggattagactgcgtc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33215 CTA TGT AGC CCA GGC TGT CC Wild type Forward A
33216 GAC GCA GTC TAA TCC CAA CC Common A
33217 Fluorophore-1 CGC CTC CCC TAC CCG Quencher-1 MUT Probe
33218 Fluorophore-2 ACA GAG ATC CTC CTG CCT CTG Quencher-2 WT Probe
oIMR5316 CTA AAG CGC ATG CTC CAG AC Mutant Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33215 0.40 uM
33216 0.40 uM
oIMR5316 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.