Protocol 21447: Probe Assay - Anapc1<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= 103 bp

Wild Type = 117 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32469 CAC TTT CTG ATG CCG TTA GC Common A
32470 Fluorophore-1 TGA TTT TAG GCA TTG TGG TGT G Quencher-1 WT Probe
32471 Fluorophore-2 TGG TGT TTG GAT TGA ACT GC Quencher-2 MUT Probe
32472 TCA GAC TTA TCC TGT GAT ATA ATG AAG Wild type Reverse A
32473 TCA TGG CTC TCT GAA AGG AAA Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32469 0.40 uM
32472 0.40 uM
32473 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.