Protocol 37818: Probe Assay - Ldhal6b<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

>chr17:5418581-5418683 103bp CACTTCCTCCACCTGAGTGC ATGCCAAAGCCCTTGAAATC

Mut = 103 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GTGGCCTCAACTGTCTTCCTCACCCTGTTCACTTCCTCCACCTGAGTGCGCAGCCATGGCCTgggctacaagcatcttgagagccagcagaagagtgagtacaaccagggtagatttcaagggctttggcatagctctatggcccagtcaggtgcaacgtgtccctcccaaccgggcctggtggagcctcacgcccacgtcctccatggcgaccgtgaagggtgaacttatccagcacatcagctccgaagagcccgttctccacaacaaggtctccattgtaggaactggatcagtcggcatggcctgtgccatcggcatcatagcgaaaggcttgactgacgagctcgcccttgttgataataatgaagaaaaaatgaagggtgagacgatggatcttcagcacggcagtgtcttcatgaaaatgccaaatattgtttgcagcaaggatttccgtgtcactgccaactctgaggtagtgattatcacagcgggtgcacgccaagagaaaaatgagacacgcctgaatttagtccagcgaaacgtaaccatctttaaggcaatggttgcggaaattatcaagcacagccctcgctgcaaaattattgttgtctccaatccagtggatatcttaactttcgtaacctggaagttgagtgggtttcccaaaaaccgtattattggaagcggctgtaacctagatacagctcgcttccgttacatgctcgggcagaggcttgggatccactctgaaagctgccacgggtgggtccttggagagcatggagattcaagtgtccctgtgtggagtggtgtgaacatcgccggcgtgccgctgagagagctgaattcagctataggaacaagcaaagaccccgagaaatggggcgatgtccacaaagaagtcattgccagtgcctataatattatcaaaatgaaaggttatacttcctgggccattggcctgtctgtaactgatattgcagagagtatcctgaagaatctcaggaagacgcatccagttaccaccaaaatccaaggtctctacggaatcaaagaggaggtgttcctcagcgtgccttgtatcctgggagagagtggcatatctgatatcataaaggtaaagctgagccctacagaggaggcccagatggtaaagagcgcagagactattTGGAAAATCCAGAAAGATATTAAGTTTTAAGTCACCCTTCTAAAGATACCGAAGACAGAAAACGCAGTGCACGGCCAAGTGTGGGGTGAAGCGGGGGTTCCG

This mutation is a 1112 bp deletion beginning at Chromosome 17 position 5,417,539 bp and ending after 5,418,650 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52227 CAC TTC CTC CAC CTG AGT GC Common A
52228 ATG CCA AAG CCC TTG AAA TC Wild type Reverse A
52229 GCA CTG CGT TTT CTG TCT TC Mutant Reverse A
52230 Fluorophore-1 CAT CTT GAG AGC CAG CAG AAG A Quencher-1 WT Probe
52231 Fluorophore-2 CCT TGG AAA ATC CAG AAA GAT ATT AAG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52227 0.40 uM
52228 0.40 uM
52229 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.