Protocol 21429: Probe Assay - Kctd1<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

Mut= 117 bp

Wt= 119 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32434 TGA AAT AAA TAG AAT TTT TCC ACA AGC Common A
32435 Fluorophore-1 CTG AGA GTC ATC AGG ACT GGA C Quencher-1 WT Probe
32436 Fluorophore-2 AGC GCT GGG CAT TAT CTG Quencher-2 MUT Probe
32437 AGA AAG AAA ACT TCA CGT GCT CAG Wild type Reverse A
32438 TGG GCT CCT GAA TAA TGG A Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32434 0.40 uM
32437 0.40 uM
32438 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.