Protocol 30105: Probe Assay - Nt5dc3<em1(IMPC)J> 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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr10:86267747+86267837 91bp CCCCTTTGGTGTCTGAAGAG ACCCTGCAGCCACTCACTAC

Mutant=  108 bp

Wild Type = 91 bp

Sequence

Wt Sequence: cccctttggtgtctgaagagtagacagccctgcgcattcatgcccagaccctgcCCttgtacagaaactctgtagtgagtggctgcagggt

 

Mutant Sequence: tgtaccactgagctgcatccccagacttggatctgcccgttctgaacattatatggatgtgcaaactgctcCCttgtacagaaactctgtagtgagtggctgcagggt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33912 CCC CTT TGG TGT CTG AAG AG Wild type Forward A
33913 ACC CTG CAG CCA CTC ACT AC Common A
33914 TGT ACC ACT GAG CTG CAT CC Mutant Forward A
33915 Fluorophore-1 TTG GAT CTG CCC GTT CTG Quencher-1 MUT Probe
33916 Fluorophore-2 CGC ATT CAT GCC CAG AC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33912 0.40 uM
33913 0.40 uM
33914 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.