Protocol 38126: Probe Assay - Rexo1<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

>chr10:80547672-80547808 137bp GGATTGCTCACGTACCCAAC TGCTCAAGGTCAGGTACAACA

Mut = 142 bp

Wt = 137 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GGTGCTGATCCTCCGTAGGGCTCTCATTGGCAGGACCGCgtagggtctggcggcaatgaggctgggtgggaaggcaggggtgtgacccaaggaggcaccagagtgaggagtgcatgtgtgttttcagccacccaaggagaaggctgaggagaagacacatgcgggcctgaccactttgttcccggggcagaagaggagggtctcacacctttgcaagccaggcaaggaggtaagacccagatatggggatacagggcgggcctctggaggaaggagatttgccaccacagttgggcaaaggacccctgctgtcggcctttggtaagctggtatctggggcttcatgtcccatccagatctgggcacaggaacagtgcacggaggggtggataaatccccatgggttttagagttgagtgggcagtccaacctcagagcccaggctatctcagttgtggccggaaggtggtgaggatagtaggggaggcctgtgtggggtgtggtagcaagcaagggctccacaaacatccctggtgcagtctccgaggagacatgagcgcagacagagtactggtgggcagaggaacatggaagaggagcttctgcagcctcctcaccatgcttgtctggagtgctctggacgggggctagctctaggatctgtgctctttcagtcagaggccccaaagaggacccccgtggctcccccagcacgccccccgactgctcaggaggtgtgctaccggcgagcacagctagcacagagggactcggccagttggctacaggccgccccgcggccaacagagaggctctcctccgtgcacatctcagcgcctggggagaagaggaggattgctcacgtacccaacccccgactggctgccggtaagtcctgaccctgccctttcttggaccccttgccttggctcgtgcagtatgagaggaagcagcgcTCGGAGAACTTATGTTGTACCTGACCTTGAGCACCCTCC

This mutation is a 917 bp deletion beginning at Chromosome 10 position 80,547,705 bp and ending after 80,548,621 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53071 TGC TCA AGG TCA GGT ACA ACA Common A
53072 GGA TTG CTC ACG TAC CCA AC Wild type Forward A
53073 GAC ACG CCC ACT TGT CCT Mutant Forward A
53074 Fluorophore-1 CGG TAA GTC CTG ACC CTG CC Quencher-1 WT Probe
53075 Fluorophore-2 CTC ATT GGC AGG ACC GCT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53071 0.40 uM
53072 0.40 uM
53073 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.