Protocol 37452: Probe Assay - Tbc1d9b<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

>chr11:50140636+50140749 114bp GGGGAAAAGGATTGTAGATTTACTG CCTCCTGCATGTTTCAGCTC

Mut = 103 bp

Wt = 114 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CTGTGGGAAGAGCACTGTGCTTCCGTTTCCACTCTCCGTGCACTGCTCAGACAGATGCTGGGGAGTGGGGGAGGCCAGCTGAAGACAGTAGCTGTTTGCCTGCCCTGCAgggactctgtcaacaacgggacaaagaaatctgataaatttacctgtttacagccgccttgtagattgtctctagaagaacaggagtactcttggtaggggagggaggactctcactgtaatagaaagtgtaaacaggagaggctcatccttgccttgggaggagggggtcacttgaatagagagagagcactggggacccagccctctgcttgagtctgagctctgatttcatccactctcactgaactaggttcttcccgaaaagagatcaccaaacactgggaatggctggaaaacaacttgcttcagacactgtccatcttcgacaacgaggaggacatcactaccttcgtcaagggcaagatacacgtaaggcccatgtcccaccgacaaccaacccagctccctttgtgaagtgctagcggccacacacagttgttgctccatatctgtgggatacaatgaattccatggattcaaccaactgtgtgtcgaaaatatttggggaaaaggattgtagatttactgaacactgtcagtctttcctaggcaatacagaagagcagagcatccctgcTGTGGTGCGTGTTGTAAGATGAGCTGAAACATGCAGGAGGGGGGACAGGCAAACTATCAACATACATAGATTTATATAT

This mutation is a 579 bp deletion beginning at Chromosome 11 position 50,140,131 bp and ending after 50,140,709 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51362 CCT CCT GCA TGT TTC AGC TC Common A
51363 GGG GAA AAG GAT TGT AGA TTT ACT G Wild type Forward A
51364 TCA GAC AGA TGC TGG GGA GT Mutant Forward A
51365 Fluorophore-1 AAT ACA GAA GAG CAG AGC ATC CC Quencher-1 WT Probe
51366 Fluorophore-2 AGG CCA GCT GAA GAC AGT AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51362 0.40 uM
51363 0.40 uM
51364 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.