Protocol 32003: Probe Assay - Bckdhb<em1(IMPC)J> - 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=  105 bp

Wild Type = 116 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32617 GCA CTG TGT AGT TTT CTC TTT ACC C Common
32618 Fluorophore-1 TAC CTG GGA TTT GCC TCT CA Quencher-1 WT Probe
32619 Fluorophore-2 CAT TTG AAG ATA CAA CGT CAT GC Quencher-2 MUT Probe
32620 TGA AAA GTA AGA AAC CTA AGC ATA CCA Wild type Reverse
32621 GAC ATG CTA CAG TGA CTC CTT CC Mutant Reverse

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
Primer 1 0.40 uM
Primer 2 0.40 uM
Primer 3 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.