Protocol 35727: Probe Assay - Txndc11<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

>chr16:11128273-11128378 106bp CCTTGTCAGGTGCAAAGC CAAGCACACATCTCCTGAAGC

Mut= 114 bp

Wt= 106 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AACTCCCCTTAATAAATTATGTTTTCTAAAAGTTGAGTCACCTGTAAATGTCTCCTATAGGAACTGTACTCATGGTGCTGTATACGGTTTCCTCAGACTTGTGAGCAATAGAGCAGTAGCAGCAGTGCCTGGAGATCAGTGGCTTTTGTTGACTGTCGGGCTACAATGTTGAAATTTTGGGTTGTGAGCGGTGAGACcgtggagcgtgtcattatttactgcttccctaacccacacctgcagggacaatgcccagcatctgtactgtcccagccaggtgttgccttagcatggtgtattggtttctgttcagggcggtgacttgcaagctcttaatttcttactcaaaagaggcctttaaaaaaagtcttctctcttcttccgtacaaaagtgtaatggaagaaaataagtaagtggagaaattgtttgtgtttactcttttttagtcgagcaaaagacgtgataataccagcaaagccgcctgtcagctttttctcctcgagatctccggtccttgacctcttccaggggcagctggactatgctgaccatgtccgccaggattctgaggtggtggtgctgttcttctatgccccgtggtgtggccagtccatcgctgccagggcagaaattgagcaggcagccagccggctttctgatcaggtaatgtgtgctttcacccaagcggtgactgtcaagtctatattagagaagatggcctctcagagaaatgatgccagactggccgtgatatccaggcaaactccggggccttgtcaggtgcaaagcttgtgaagaaacaggaacaagaatcctggcatgaggtcAAGTGGCAAGAGTAAGCTTCAGTTCTGTAGCTTCAGGAGATGTGTGCTTGCCCAGGATACCAGGTTGTGGGATACAGTGCTGAAGATAGTAGAGACCAAGAACCCCAGCTCTCTCATGTCCAGCTTATGTGGCTTGTAAGCCTTTTATGTTGTAAGTGGCACACAGGCCATGGTGAATGTG

This mutation is a 631 bp deletion beginning at Chromosome 16 position 11,128,323 bp and ending after 11,128,953 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46870 CCT TGT CAG GTG CAA AGC Wild type Forward A
46871 CAA GCA CAC ATC TCC TGA AGC Common A
46872 GAT CAG TGG CTT TTG TTG ACT G Mutant Forward A
46873 Fluorophore-1 ACA AGA ATC CTG GCA TGA GGT Quencher-1 WT Probe
46874 Fluorophore-2 TGA GCG GTG AGA CAA GTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46870 0.40 uM
46871 0.40 uM
46872 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.