Protocol 45423: Probe Assay - Mmp25<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

>chr17:23644372-23644461 90bp GGCTCTTGGAGCTCTGGAAT CCCAGCTTCCCATTTTAGC

Mutant=  111 bp

Wild Type = 90 bp

Sequence

Wt Sequence: ggctcttggagctctggaatactgtcTctatcaggaggtttggtggctgagattcagaagtttgaaaaggagctaaaatgggaagctggg

MUT Sequence: ggctcttggagctctggaatactgtcTCtaaattgcaagaagttaaagatgttggtctcaggttccagcccagaccttggactctcactggccagtctttgtcaggctcca

A 638 bp deletion beginning at Chromosome 17 position 23643797 bp and ending after 23644434 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65577 GGC TCT TGG AGC TCT GGA AT Common A
65578 CCC AGC TTC CCA TTT TAG C Wild type Reverse A
65579 TGG AGC CTG ACA AAG ACT GG Mutant Reverse A
65580 Fluorophore-1 TGT CTC TAA ATT GCA AGA AGT TAA AGA TG Quencher-1 MUT Probe
65581 Fluorophore-2 CTA TCA GGA GGT TTG GTG GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65577 0.40 uM
65578 0.40 uM
65579 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.