Protocol 21313: Probe Assay - Hmcn1<em1J> 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

Mut=110 bp

Wt= 125 bp

Sequence

WT Sequence: tggctggctctcttcatttttataatctgaaagaagtatttgttgtctctcaagggactcaagaaatgtcatTTTCTTTCCCCACCCATGTCtagaatgaattacgcaatagatacagaatatga

MUT Sequence: acataattgaaagtgcccatatacagatactatacacctcattgagaactcatccacatatttgagtaatagatctagaatgaattacgcaatagatacagaatatga

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32192 ACA TAA TTG AAA GTG CCC ATA TAC AG Mutant Forward A
32193 Fluorophore-1 CAC CTC ATT GAG AAC TCA TCC A Quencher-1 MUT Probe
32194 TGG CTG GCT CTC TTC ATT TT Wild type Forward A
32195 Fluorophore-2 TCT TTC CCC ACC CAT GTC TA Quencher-2 WT Probe
32196 TCA TAT TCT GTA TCT ATT GCG TAA TTC Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32192 0.40 uM
32194 0.40 uM
32196 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.