Protocol 45433: Probe Assay - Mybph<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

>chr1:134196854+134196954 101bp AATGGAAGGCTTTGTTCTCC GAGAAAGCAAGTGTGGGTTCC

Mutant=  107 bp

Wild Type = 101 bp

The common primer (primer 65605) anneals over the nucleotide sequence containing mouse genomic variations rs30878889.

Sequence

Wt Sequence: aatggaaggctttgttctcccggtgaagaggcagctcgagctgtaggaacgtcgtAggtaggagctgtgatgtctagcccggaacccacacttgctttctc

Mut Sequence: aatggaaggctttgttctcccggtgaagaggcagctcgagctgtaggaacgtcgtATcggggctcaaaaggttcatcctgctaagaggctctggatgaaagtccagg

A 1002 bp deletion beginning at Chromosome 1 position 134,124,648 bp and ending after 134,125,649 bp.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65605 AAT GGA AGG CTT TGT TCT CC Common A
65606 GAG AAA GCA AGT GTG GGT TCC Wild type Reverse A
65607 CCT GGA CTT TCA TCC AGA GC Mutant Reverse A
65608 Fluorophore-1 AAC GTC GTA TCG GGG CTC Quencher-1 MUT Probe
65609 Fluorophore-2 TAG GTA GGA GCT GTG ATG TCT AGC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65605 0.40 uM
65606 0.40 uM
65607 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.