Protocol 21493: Probe Assay - Fbxo10<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=  98 bp

Wild Type = 112 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32578 GAA GGC AGC TGC ATA GCT TAG Common A
32579 Fluorophore-1 GTT TTG TCC TGG CTG TGT CC Quencher-1 WT Probe
32580 TGC TTG AGT ACT GCC TCA CTG Wild type Reverse A
32581 Fluorophore-2 AAT TGC TCC TCG GGA GAT TAC Quencher-2 MUT Probe
32582 TCG TCA GGT TTG GTA GCA AGT Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32578 0.40 uM
32580 0.40 uM
32582 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.