Protocol 45866: Probe Assay - Lrrc40<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

chr3:158041717+158041823 107bp CTTGAACTACAGTGTGGCCCAGTA TGGGATGATGTAAAGATCTGGTC

Mutant=  110 bp

Wild Type = 107 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

ATCTGTTCTGTGTGTGATATTAAGAGACCACCAGAACTCTGCTGCTGTTGACGCCTCCTCAAGTCAAGTCTGTGTTTGTTTCTTTTCATTTTAGTAATGACTTATTTGTTCACAATTTCTGGACAGCAgtttggtcaatatacacagaataaattactaatcacccaacacattgccatttaatatgaaaaacaggagtttagtaaattattttttttcaatggcaaagctcaactattgctaattgtaagactaagaatattgactttgtcttgtaatcattttagatacatgataatcagctgacatctcttccttcagctataagagagctagacaatcttcagaaacttaatgtcaggtaaagtgtgaacctgtttctaaaactgaaatcccttttcttcacatagaaaataaccttgaactacagtgtggcccagtaggaatcaggcttggttaaacacctgccagatgctgtattctaccCAAGAGGAGAAAACAAGACCAGATCTTTACATCATCCCAGAGAATTTAATGAATCAATTATTTGTTTTC

 

This mutation is a 358 bp deletion beginning at Chromosome 3 position 158,041,427 bp and ending after 158,041,784 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66656 TGG GAT GAT GTA AAG ATC TGG TC Common A
66657 CTT GAA CTA CAG TGT GGC CCA GTA Wild type Forward A
66658 CTC AAG TCA AGT CTG TGT TTG TTT C Mutant Forward A
66659 Fluorophore-1 ACA ATT TCT GGA CAG CAC AAG AGG AG Quencher-1 MUT Probe
66660 Fluorophore-2 TCA GGC TTG GTT AAA CAC CTG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66656 0.40 uM
66657 0.40 uM
66658 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.