Protocol 30116: Probe Assay - Kash5<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

>chr7:52451576-52451673 98bp AACTCAGGAGAGGGACTTGC AGGCCACGGTTTAAGAACAG

Mutant=  96 bp

Wild Type = 98 bp

Sequence

Wt Sequence: aactcaggagagggacttgccccaggtggctctgcatgctgtagggcaggcgtgtcatCAacctgctgatgacagacactgttcttaaaccgtggcct

 

Mutant Sequence: aactcaggagagggacttgccccaggtggctctgcatgctgtagggcaggcgtgtcatCTgagaaccagctgccttgggtgatggtgcaggggtcc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33945 AAC TCA GGA GAG GGA CTT GC Common A
33946 GGA CCC CTG CAC CAT CAC Mutant Reverse A
33947 AGG CCA CGG TTT AAG AAC AG Wild type Reverse A
33948 Fluorophore-1 CTG AGA ACC AGC TGC CTT G Quencher-1 MUT Probe
33949 Fluorophore-2 TCA TCA ACC TGC TGA TGA CAG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33945 0.40 uM
33946 0.40 uM
33947 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.