Protocol 33537: Probe Assay - Ttc7b<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

>chr12:100499929-100500066 138bp GCCACAGGGACCAAGAAGT CCGAGGGTGGATACACTGA

Mut= 152 bp

Wt= 138 bp

Fam=Mut

Hex=Wt

Sequence

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

CTTCTCTTGCAAATAGACAGCATTGGACTTTATGTGTATTTCACAAGATCATCATATGTTTTACCATTACAGGAGAATTTCTTTACTCCCTTCTGATCTTAAATTTCCAAATTAATATGTAACCATCTTGGAGTCCA^taactatgtggcaaagaaatgacttagcgcttgagccaacactgctggaatgttccctgctaaagagcactctctcacatagacacaatcttgggttctaaaggttccagaagagcctcgccagaaacccggaagcttagtgcatgtggggcgtgaggtcctcggggcgtgaggtgctcagggctttcctgcacacagaccgtttcttattcatttatcgtctcacttggttctgcagatgatatggctgaacttctccttggggagtcgaaactggaacagcacttgaaggagaagcccctgcggcagggagccagtccccggggccccagaccccagctgactgaagtgcgcaagcatctgaccgctgccctggaccgagggaacctcaaggtactgtgtgccacagggaccaagaagtgatggtagtgggtactgccatcatctcctagggacagtgtggtgagcaaggtgtgtttggcgcacacgtgtcttctttagcaggcacacgccctgcct^GATCAGTGTATCCACCCTCGGCTGGCTGGCCTGTGGGGCTCCCAGCCTAGTAGGAGTCTCTGATGGGAGTCTGCAATAACACATTCAACAGAACGAGGGGTTCATTTCCCCCCAGGCACTT

This mutation is a 519 bp deletion beginning at Chromosome 12 position 100,499,950 bp and ending after 100,500,468 bp (GRCm38/mm10).  There is also a single bp insertion (A) at the deletion site that will not alter the results of the deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41640 GCC ACA GGG ACC AAG AAG T Wild type Forward A
41641 CCG AGG GTG GAT ACA CTG A Common A
41642 TGC AAA TAG ACA GCA TTG GA Mutant Forward A
41643 Fluorophore-1 TAG CAG GCA CAC GCC C Quencher-1 WT Probe
41644 Fluorophore-2 TGT AAC CAT CTT GGA GTC CAA GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41640 0.40 uM
41641 0.40 uM
41642 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.