Protocol 21346: Probe Assay - Pdzrn3<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= 108 bp

Wild Type = 118 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32252 GGT CAA ACT CAG CCT CTT GTG Common A
32253 Fluorophore-1 CCC AGA GGC AGG ATG TTA GT Quencher-1 WT Probe
32254 Fluorophore-2 TTC CAA CTG AGA AAC TGT GGT C Quencher-2 MUT Probe
32255 GAC TCC TCC GGC CGT ATT G Wild type Reverse A
32317 TCA CCC TAT ATG TCC TGC CTT T Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32252 0.40 uM
32255 0.40 uM
32317 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.