Protocol 30757: Probe Assay - Zmynd19<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=  137 bp

Wild Type = 148 bp

>chr2:24952437+24952584 148bp GTTGTTGATCAGGATGTGACC TGAGAAGAAGCACAGAGCAGAG

Sequence

Wt Sequence: gttgttgatcaggatgtgacccctGtacttttggggtcctaggagcaggatatcatctgtacttctggcatttttctgagctggatttgaaatatgctgagagttaaaggctttccaacagatgcactctgctctgtgcttcttctca

 

Mutant Sequence: gttgttgatcaggatgtgacccctGagaaggatccatgggtctcatctcatcaaaatcccactagttgtttaaagtgatatgtacattagtcactcctttgGgtgagtcataggttacatccttagaaacggggtca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35386 GTT GTT GAT CAG GAT GTG ACC Common A
35387 TGA CCC CGT TTC TAA GGA TG Mutant Reverse A
35388 TGA GAA GAA GCA CAG AGC AGA G Wild type Reverse A
35389 Fluorophore-1 ACT CCT TTG GGT GAG TCA TAG GT Quencher-1 MUT Probe
35390 Fluorophore-2 TTT TGG GGT CCT AGG AGC A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35386 0.40 uM
35387 0.40 uM
35388 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.