Protocol 33176: Probe Assay - Trpa1<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

>chr1:14910565-14910688 124bp AGGCCAGAATCCACATCTGA CGTCACTACCACCACCATCA

Mut= 111 bp

Wt= 124 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTATCTTTTGGGCAATTGCAAGCTAATTATAAATATAAGTCACATGGAACCTAATTATGATTTGCTATAATGTAATCTCAGCATTATAGAACAATTATGAAATTAATATGCAGATTAAAAGTATAGTAACAAGAGGATCTGTTTCTATAAATACTATGTAAAAAATTAACCCAGTGTTTGGATCTATAGGTACTTTCACCTGGTCTCAAGTGGTGTCCATTGGgtaattctgaagctaaaactacatcctatttctatagttcaagaaagtgtacctggttttataagcacttgactccgaggttcggagtgggtagttcttcaggatatattaagtaaatcccaaacaacatttaatgcatattttgttatttctttagtgctgaatataatggatggttatggaaataccccactgcattgtgctgcagaaaaaaatcaagttgaaagtgtaaagtttcttctcagccaaggagcaaatccaaacctccgaaatagaaacatgatgtcaccccttcacatagctgtgcatggcatgtacaacgaagtgatcaaggtgagcctgctggccatcacagtgaggctgactgtggagctaggccagaatccacatctgactcacttactgtttcaaagtgactgacttcaagtgccatgtcatggCAAATGAAACGTAACTATTATGTTTATTTTTCGCCAAATGATGGTGGTGGTAGTGACGATATGACGGCAATGGGGAAGATGATGATGGATAGCACTGATGAGAAAGATGGTGGTAACGACTGTCATAGTAACCAGAGTTAACTTTTCTTGAGTTTTCCTTTATCAGGTATTTTAAAGAAGAGCTGTGTGACTTTATTAAGTTACCATTT

This mutation is a 440 bp deletion beginning at Chromosome 1 position 14,910,623 bp and ending after 14,911,062 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40826 AGG CCA GAA TCC ACA TCT GA Wild type Forward A
40827 CGT CAC TAC CAC CAC CAT CA Common A
40828 CCA GTG TTT GGA TCT ATA GGT ACT TTC Mutant Forward A
40829 Fluorophore-1 CTG TTT CAA AGT GAC TGA CTT CAA GT Quencher-1 WT Probe
40830 Fluorophore-2 CCA TTG GCA AAT GAA ACG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40826 0.40 uM
40827 0.40 uM
40828 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.