Protocol 30346: Probe Assay - Dgkb<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= 109 bp

Wt= 105 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34381 AGG TAA AAG TTT ATG TCA TGT GGT TG Common A
34382 Fluorophore-1 CAT GGA GAT AGA AAC ATC TAA CTA GGA Quencher-1 WT Probe
34383 Fluorophore-2 TTG ACA TAG AAG TTC ACA TTA TCT TGA Quencher-2 MUT Probe
34384 ATG ACA GGC ATG TGT CTC ACC Wild type Reverse A
34385 TTC CAA ACT TGA TGA AAA TGT CTC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34381 0.40 uM
34384 0.40 uM
34385 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.