Protocol 38028: Probe Assay - Tifab<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

>chr13:56176178-56176268 91bp TCCCTTGAGACCTTTGTCTGC TCCCGGTTCATTCATCTGTC

Mut= 99 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case;and insertions with carrots ^g^ (ggctg insertion):

CTTTATTCCTTGCTGAGAAGGAAGGGCACACCTGTGTTATAAAACAATACAATTTGGAGAGGATGGGCAGTTATGTCCTGTTGGAGTATAGGGAGATGGAATTCCAAGTTTAGTGTCAACCTCCACCCCATCCCAGCTTGGGGAGAATAACTGTATCTATTTACACATGGTCCTCATCAGCAAGATGTGGCCCCAAGATCTCTAAGTAGGCAGCTTGCCTGGGCTCAGAAAACTACAGAAGTGTGTCTTTGTCATGTTCTTCCCAGGATCTATGGAGAGGCCCCTCACAGTCCTGCAGGTGAGCCTGTACCACCCTA^cacagggcccggtcgcctttgcccacgtcccacagcagctgcaacatgacgccagccggctgttggttgggcgaggacagaacacccatctccagctgcagctgccacagttgtctcgctaccatttgtccctggaaccctacctggagaaaggcagcagtctgctggccttctgcctcaaggtgctgacccgcaagagctgtgtgtgggtcaacgggctgccactgaggtacctggagcaggttcccttgggcaccatcaacagaatctccttctctggcatccagatgctagtccgcaaagaggggggagcctcccttgagacctttgtctgctacttccatctcagcccttcacccctgatttac^AGACCCAAGGCTCAGGAGACAGATGAATGAACCGGGAAAACTCTGTTAAGAAAGAGACTCCCCCTGTTTTGGGGGAGGCAACTCATGAACTCTCGAGGTCTCTCCACAGCCCCTCCCAGTCTCAGACAGCTGCTTCCCTGCCAAGCCCGGAAGAAGCAATAGAAACAAAGTTGCAGAAGGGGCCCTCAGACAATGCACCCCACTAGCCCTGCTGCTTGAGAGCAAACCTGACAAAAATAGGGTTTGAGATGGTAGAGCTATATCCTGTGAGGTTGACACACAGAGTTCACATGTAAAGCCTGCTGGCCGCCATGTTCAC

This mutation is a 368 bp deletion beginning at Chromosome 13 position 56,176,215 bp and ending after 56,176,582 bp (GRCm38/mm10). In addition, there is a 5 bp insertion (GGCTG) at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52824 TCC CTT GAG ACC TTT GTC TGC Wild type Forward A
52825 TCC CGG TTC ATT CAT CTG TC Common A
52826 TCC CAG GAT CTA TGG AGA GG Mutant Forward A
52827 Fluorophore-1 TCA GCC CTT CAC CCC TGA Quencher-1 WT Probe
52828 Fluorophore-2 ACC ACC CTA GGC TGA GAC CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52824 0.40 uM
52825 0.40 uM
52826 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.