Protocol 21106: Probe Assay - Chd3<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

Mutant=  118 bp

Wild Type = 114 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31767 AAC ACT GTT TGC ATC CGT TG Mutant Forward A
31768 GGG TTC CTA GGG TCA AGA GA Wild type Forward A
31769 ACT GAC AAC TGG ACT CTG TAC CA Common A
31770 Fluorophore-1 AGG AGT TAT TGC CTT GGT TCC Quencher-1 MUT Probe
31771 Fluorophore-2 CTT GGA CTT GGA ACC CTA ACC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31767 0.40 uM
31768 0.40 uM
31769 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.