Protocol 21516: Probe Assay - Kynu<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= 113 bp

Wt= 119 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32640 CCT TGG CTA TTT GTG ATT GG Mutant Forward A
32642 Fluorophore-1 AGC ATA GTA ACT GCG TGA GAT CG Quencher-1 MUT Probe
32643 GAA ATT CCC TTG GCC TTC A Wild type Forward A
32644 Fluorophore-2 AGT GGG CCA AGA TGT AAG TAC C Quencher-2 WT Probe
32645 GAA TTC TTC TGT CAG ATG GAG TTA CA Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32640 0.40 uM
32643 0.40 uM
32645 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.