Stock No: 017537
Protocol 32721: Probe Assay - Tmem173<gt> Probe
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

>chr18:35735985-35736089 105bp TCAGCTACATAACAACATGCTCAG TTGGGGTCAACTACACTCAGG

Mutant=  105 bp

Wild Type = 105 bp

Sequence

Wt Sequence: TCAGCTACATAACAACATGCTCAGTGGTGCAGGGAGCCGAAGACTGTACAtCCTCTTTCCATTGGACTGTGGGGTGCCTGACAACCTGAGTGTAGTTGACCCCAA

 

Mutant Sequence: TCAGCTACATAACAACATGCTCAGTGGTGCAGGGAGCCGAAGACTGTACAaCCTCTTTCCATTGGACTGTGGGGTGCCTGACAACCTGAGTGTAGTTGACCCCAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39817 TCA GCT ACA TAA CAA CAT GCT CAG Forward A
39818 TTG GGG TCA ACT ACA CTC AGG Reverse A
39819 Fluorophore-1 CGA AGA CTG TAC ATC CTC TTT CCA Quencher-1 WT Probe
39820 Fluorophore-2 CGA AGA CTG TAC AAC CTC TTT CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39817 0.40 uM
39818 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.