Protocol 21622: Probe Assay - Lpgat1<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

Mut= 115 bp

Wt= 112 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32859 TGG TAC AAT AGT TTA GGG GAA TTT G Common A
32860 Fluorophore-1 TCT GAA TTC TTT CCC ATG ATA TCT TTA Quencher-1 WT Probe
32861 Fluorophore-2 TAA ATT TTG AAT GTC TGG TGT TGA Quencher-2 MUT Probe
32862 ACA ACT TAG ATG TTT ACC AAC AGG AA Wild type Reverse A
32863 CAA AAT CAA CAG TTC AAA CAC TGG Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32859 0.40 uM
32862 0.40 uM
32863 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 4.0 -- 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.