Protocol 21666: Probe Assay - Tg(Itgax-cre,-EGFP)4097Ach 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=  80 bp

Wild Type = 134 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32968 Fluorophore-1 CAC TGG GGA GCA TTT ACC AG Quencher-1 Internal Positive Control Forward A
32971 Fluorophore-2 GTG GTC AAG TTT CAT CTC AGC A Quencher-2 Internal Positive Control Reverse A
32975 TCA GAG CCT GCT TCT GTT CTC Transgene Forward A Itgax
32976 CAA TCA AGG GTC CCC AAA C Transgene Reverse A beta-globin
32977 Fluorophore-3 TGC AGG AAT TGA TCC TGA GAA CTT C Quencher-3 Tg Probe
32981 Fluorophore-4 CCA TGA TTC TGG GTG CAG ACA G Quencher-4 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32968 0.40 uM
32971 0.40 uM
32975 0.40 uM
32976 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.