Protocol 37720: Probe Assay - Taar8a<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

>chr10:24077389+24077519 131bp GTTGCTGGGGTACCTATTATAACTC ATTTGCAGTTGATGAATGTCC

Mut = 169 bp

Wt = 131 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TTCATTTTTTATAATTAAAATACAAAAAGGATTTAtaaccgtgggttatatttaaaattaattcttcagtatggagttcatttctttaatttttatatctcaaaattactgttgtcttctagcaggacagaaatcccccaagcatcaaaaagcagattcatgaccagcaacttttcccaaccagccctgcagctctgctatgagaacacgaatggatcctgtattaaaactccctattctccagggccccgggtcatcctgtacatggtctatggctttggggctgtgctggcagtgtgtgggaacctcctggtggtgatttcagttctccatttcaagcagctgcactctccagccaattttctcatcgcctctctggccagtgctgacttcttggtgggcatctctgtgatgcccttcagcatggtcaggtccattgagagctgctggtactttggagatgcattttgtagccttcacagttgttgtgatgtggctttttgttactcttctgtcctccacctctgcttcatctcagtggacaggtacatcgctgtcacggaccctctggtctatcccaccaagttcacagtgtctgtgtctggaatttgcatcagcatctcctggattctgcccctggtgtacagcagtgcagtgttttacacaggcatcagtgctaaggggattgaaagcttagtaagtgctctaaattgtgtagggggctgccaaattgttatcaatcaagactttgttttgataagttttcttctattcttcatacctacccttgttatgatcattctttacagcaaaatatttttggtagccaaacagcaagctgtaaaaattgaaacttctgtaagtggcaacagaggtgaatcatcctcagagagtcacaaagccagagtggccaagagagagaggaaggctgcaaaaaccttgggggtcactgtggtggcttttatggtctcgtggctcccatacacaattgacgcattggttgatgctttcatgggcttcatcactcctgcctatgtctatgaaatctgttgctggggtacctattataactcagccatgaaccctttgatttatgctttcttttttccttggtttaagaaagccataaagcttattttaagtggggaaattctaaagggacattcatcaactgcaaatttattttcagagtaagcattaatacaaaattcagaaaattccttaaggtataaaattatgaaatggtgacagaactaaatctaataaatgaccttTATGAAGCTAGAAAAATACAGCATAAAAACATTAGTCAGGAGGA

This mutation is a 1239 bp deletion beginning at Chromosome 10 position 24,076,376 bp and ending after 24,077,614 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51999 GTT GCT GGG GTA CCT ATT ATA ACT C Wild type Forward A
52000 ATT TGC AGT TGA TGA ATG TCC Wild type Reverse A
52001 CAG TGA CGT TTA ACA TAG TTA TAG GAA Mutant Forward A
52002 TTG ACA AGA CAT TCC TCC TGA Mutant Reverse A
52003 Fluorophore-1 CCT TTG ATT TAT GCT TTC TTT TTT CC Quencher-1 WT Probe
52004 Fluorophore-2 ATT GTG TGA CCA TCA ATG GGA AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51999 0.40 uM
52000 0.40 uM
52001 0.40 uM
52002 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.