Protocol 51905: Probe Assay - Zfp653<em1(IMPC)J>
Version 1.0

Notes

Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.

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=  96bp

Wild Type = 108bp

>chr9:22067839-22067946 108bp TTTGGGTCTACATCATGCC CATAGGATGAGTTTGAGGCT

Sequence

Large deletion:  Mutant sequence with junction in uppercase

agtgctggggagacaggcgtgcaccacctctcctgcatctgtaacgtttctaatgattgacacaaaggctgactacACtggtggcagtgagccctgctctgcctcttttttgatgcattagcctcaaactcatcctatggcagaggctgcctgtatc

This mutation is a 554 bp deletion of Chr9:22,067,901-22,068,454 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
80556 TTT GGG TCT ACA TCA TGC C Wild type Forward A
80557 CAT AGG ATG AGT TTG AGG CT Common A
80558 ACG TTT CTA ATG ATT GAC ACA Mutant Forward A
80559 Fluorophore-1 ATC ATG GTG GTT CAA GAG AG Quencher-1 WT Probe
80560 Fluorophore-2 CTG ACT ACA CTG GTG GCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
80556 0.40 uM
80557 0.40 uM
80558 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.