Protocol 21095: Probe Assay - Prpf40a<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= 120 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31795 Fluorophore-1 TTG CTG TTT GGT TTG GTT TG Quencher-1 MUT Probe
31796 CGA CTC CAG TGT TGA TGA GC Common A
31797 Fluorophore-2 GCA AGC CTA GGA GTT GAA ACC Quencher-2 WT Probe
31798 AAC AAT GCA ATG TAA ATC AGT TTC Wild type Reverse A
31799 GTT AAC CAT TTA AGA GTG AGG ATG G Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31796 0.40 uM
31798 0.40 uM
31799 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.