Protocol 30882: Probe Assay - Eid2<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

>chr7:28268554+28268655 102bp CCAGAGGGTGGATTTTGACA TGGATGAACTTATCGCAACC

Mutant=  91 bp

Wild Type = 102 bp

Sequence

Wt Sequence: CCAGAGGGTGGATTTTGACATTTTAACGTTTACCATAGCCCTGACTGCATCAGAGGTGATCAATCCTCTAATAGAAGaactgggttgcgataagttcatcca

 

Mutant Sequence: gttcttcccgggtctcagcgagcagcggtgctgaccccgcggtccgacatgtcgcagctgcccgcAAactgggttgcgataagttcatcca

658 bp deletion beginning at Chromosome 7 positive strand position 28267973 bp GTCAGCAGCGCCCCGCAGAC, and ending after GATCAATCCTCTAATAGAAG at 28268630 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35739 GTT CTT CCC GGG TCT CAG C Mutant Forward A
35740 TGG ATG AAC TTA TCG CAA CC Common A
35741 CCA GAG GGT GGA TTT TGA CA Wild type Forward A
35742 Fluorophore-1 ACG TTT ACC ATA GCC CTG ACT G Quencher-1 WT Probe
35743 Fluorophore-2 GGT CCG ACA TGT CGC AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35739 0.40 uM
35740 0.40 uM
35741 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.