Protocol 43455: Probe Assay - Irgq<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

  
>chr7:24531128+24531224 97bp GGACTGAAGCCTGGATCTACA TCTGTCTCCCAGAGTGCTGA

Mutant=  98 bp

Wild Type = 97 bp

Sequence

Large deletion:  Mutant sequence with junction in uppercase

ggactgaagcctggatctacagtgaatcagttagGGtagacaagcacatggatgccctctctcagtctggctctggattttagccacagggacgacagcaaatgtgatgca

 

This mutation is a 7630 bp deletion beginning at Chromosome 7 position 24,531,163 bp and ending after 24,538,792 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
63132 GGA CTG AAG CCT GGA TCT ACA Common A
63133 TCT GTC TCC CAG AGT GCT GA Wild type Reverse A
63134 TGC ATC ACA TTT GCT GTC GT Mutant Reverse A
63135 Fluorophore-1 ATC AGT TAG GAG AGG GTG TTG ATT G Quencher-1 WT Probe
63136 Fluorophore-2 TGA ATC AGT TAG GGT AGA CAA GCA CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
63132 0.40 uM
63133 0.40 uM
63134 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.