Stock No: 029254
Protocol 32441: Probe Assay - Huwe1<tm1.1Mak> 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

Mutant=  111 bp

Wild Type = 77 bp

>chrX:148364217+148364293 77bp GCAGACAAGGATGTCTTTAATGC TGCAGGCAAATTTCTATTCC

Sequence

Wt Sequence: tgctgacttagtcaccaagttttaggcagacaaggatgtctttaatgcctttgcagtgcaagcTTaatatattattaggctaggaatagaaatttgcctgcaaagcagattggtaaccctgggtttgggaggcaccatgtgcttgagtgccaccccccactccacccccagtcagtaattgttc

 

Mutant Sequence:CTGCTGACTTAGTCACCAAGTTTTAGGCAGACAAGGATGTCTTTAATGCCTTTgcAGTGCggatccataacttcgtatagcatacattatacgaagttaTAATATATTATTAGGCTAGGAATAGAAATTTGCCTGCAAAGCAGATTGGTAACCCTGGGTTTGGGAGGCACCATGTGCTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39118 GCA GAC AAG GAT GTC TTT AAT GC Forward A
39119 TGC AGG CAA ATT TCT ATT CC Reverse A
39120 Fluorophore-1 CAG TGC GGA TCC ATA ACT TCG TAT Quencher-1 MUT Probe
39121 Fluorophore-2 TGC AGT GCA AGC TTA ATA TAT TAT TAG GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39118 0.40 uM
39119 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.