Protocol 32601: Probe Assay - Ttc7<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

>chr17:87290576+87290663 88bp GCTATCCACTGGGTTCCAGA GTTGCCTCTCCCAGGTCTCA

Mut= 86 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

ATGTGAGAGAATTGAAAATGACGCAGGAAACCGTACATGCCACATTCACTCTTTGGTTTCTAGGTCGTCCTGAGCAACATTAAAGTCCCTTAAGAGGAGACAAATTGACTACACAATCTCACAAACTAGGAGACTGTGAAACGGTCGATGAAGCCCTCACAATACGGTTCTCCTCTTCCgtggtttgagtgggtgggtgctttttgttttccaatgggttcttgctctgtcacccggactgagtgagaactctcttccgtcctcagagacgatttaggtttatggagaatgcacctttatcgtctcagacttgagccattcgtgcgctgggcagggatggctcggggctcaggatatctacagcgcatgttcaaggagtcaggaatccttgtcctgtttttccagatgactttgtgaaattgctgctagcggaggccctcctggagcagtgtctgaaggataaccacgacaaaataaaaaactccatccctttgctggagaagactgatcacaggctgaatgaagccaaggaccacctgagcagcctcctcaacaatgggaaactgccggtgagccttcaggttgtctcactcagaggtcagggcagagcggggatctctaggcagcaggtggattcttgtgtatcccaagtagctccagtggatatggagaagaggcgggaaaggcagtcccaagataaaaatcagaatcctacaatctgagtgattcctgaagtcccagctagctcagctctgtggagatcagtgacgtgaagccgctgccctagcttctgctatccactgggttccagaactgcagcatctcattaaggcttcctaggtacTTTTTAGCCCCATGTCTGAGACCTGGGAGAGGCAACCACATAGCCAAATTGGTTTCACCTGCTGTGGTGTCTCAAGCTCTTGAGATGCCCAAAGAACTGGAGTGGGAATAAAATAATGACCAAGGAACTTGGGCCAGTGGAAGGACAGCTTCTAGGGGTTTCGCGATCAGAGT

 

This mutation is a 665 bp deletion beginning at Chromosome 17 position 87289963 bp and ending after 87290627 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39513 GCT ATC CAC TGG GTT CCA GA Wild type Forward A
39514 GTT GCC TCT CCC AGG TCT CA Common A
39515 GAG ACT GTG AAA CGG TCG ATG Mutant Forward A
39516 Fluorophore-1 TGC AGC ATC TCA TTA AGG CTT Quencher-1 WT Probe
39517 Fluorophore-2 TCT TCC TTT TTA GCC CCA TGT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39513 0.40 uM
39514 0.40 uM
39515 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.