Stock No: 003684
Protocol 32162: Probe Assay - Mfrp<rd6> 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

>chr9:44102579+44102694 116bp ACTACCACCCCAGCAAGGAC CTTTCCTCCCCAACACCATC

Mutant=  112 bp

Wild Type = 116 bp

Sequence

Wt Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTaagtCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG

 

Mutant Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38489 ACT ACC ACC CCA GCA AGG AC Forward A
38490 CTT TCC TCC CCA ACA CCA TC Reverse A
38491 Fluorophore-1 CAG ACC AGT AAG TCC CAA GGG Quencher-1 WT Probe
38492 Fluorophore-2 CAG ACC AGT CCC AAG GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38489 0.40 uM
38490 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.