Protocol 21347: Probe Assay - Mgme1<em1J> 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=  119 bp

Wild Type = 115 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32256 CAC ATG AGA CTG AAG AAA GAG AAT C Mutant Forward A
32257 Fluorophore-1 CAA GGC CAG CCT ATA ACA CAA Quencher-1 MUT Probe
32258 GGC TGC TGG ACT GTG TGG Wild type Forward A
32259 Fluorophore-2 TAC ATA CCC GTG CCT CCT TC Quencher-2 WT Probe
32260 AAG AGA CAG CAG CCC CAT A Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32256 0.40 uM
32258 0.40 uM
32260 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.