Stock No: 006335
Protocol 32572: Probe Assay - Mgat5<tm1Jwd> 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=  104 bp

Wild Type = 133 bp

>chr1:127306714+127306846 133bp ATGAACTTGCCTTCCCCTTC ATGAAGCCGAAAGTCACCAG

Sequence

Wt Sequence: ATGAACTTGCCTTCCCCTTCTGCTTGTTGAGAGCCAagGGAATGGTACATTGCTAGAGAGAGATGGCTTTCTTTTCTCCCTGGAAGTTGTCCTCTCAGAAGCTGGGCTTTTTCCTGGTGACTTTCGGCTTCAT

 

Mutant Sequence: ATGAACTTGCCTTCCCCTTCTGCTTGTTGAGAGCCAAAGCTTATCGAGCTTGCATGCCTGCAGGTCGGAGGCCACCATGGCTCGCGATGATCCCGTCGTTTTAC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39432 ATG AAC TTG CCT TCC CCT TC Common A
39433 ATG AAG CCG AAA GTC ACC AG Wild type Reverse A
39434 GTA AAA CGA CGG GAT CAT CG Mutant Reverse A
39435 Fluorophore-1 TGA GAG CCA AGG GAA TGG TA Quencher-1 WT Probe
39436 Fluorophore-2 TTG CAT GCC TGC AGG TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39432 0.40 uM
39433 0.40 uM
39434 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.