Protocol 35404: Probe Assay - Rufy1<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

>chr11:50420446-50420573 128bp GCTTTTGAAAGGAAGCCAGT TCCAAAGGACCAAAGAAAGACT

Mutant=  108 bp

Wild Type = 128 bp

Sequence

GCTTTTGAAAGGAAGCCAGTGGGGCCCAGGAGGGCCTGCAGCCCATTctcatgacttacactcatgtttccccttttagtcaagaagagtttcattggccaaaataagtctttctttggtcctttggagctggtggaaaaactttgtcctgaagcatcagatatagctaccagtgtcaggaatcttccagaactaaagtgagtaagaagtagtaacgctgagtgtttgcttgcctctctcgggtgtttgccacctCTGAAGTCCTTCCTAACTTAACACTCTGTAGCAGGAACCATGACATGGATTCTGACCCCACCTGTGCTGCTCACTCGTCACATGACCTTGAGCGAGACTT

A 208 bp deletion beginning at Chromosome 11 position 50,420,319 bp and ending after 50,420,526 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45972 GCT TTT GAA AGG AAG CCA GT Common A
45973 TCC AAA GGA CCA AAG AAA GAC T Wild type Reverse A
45974 GTG GGG TCA GAA TCC ATG TC Mutant Reverse A
45977 Fluorophore-1 TCA TGA CTT ACA CTC ATG TTT CCC Quencher-1 WT Probe
45978 Fluorophore-2 CAG CCC ATT CTG AAG TCC TTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45972 0.40 uM
45973 0.40 uM
45974 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.