Protocol 29921: Probe Assay - Alg8<em1J> Probe Alt 2
Version 2.0

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=  133 bp

Wild Type = 137 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32382 AAA GTG TTT TCT AGA TTA TGG ATT TTG Common A
32383 Fluorophore-1 TTG CCT GAG AAC CTG TTT GA Quencher-1 WT Probe
32385 GTT TGT TTG TGA ATG GGT CTC A Wild type Reverse A
32386 ATC TTC CTG GCC TAG AGT GG Mutant Reverse A
33662 Fluorophore-2 TGT GCA CAA AGT CCT AAG GTG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32382 0.40 uM
32385 0.40 uM
32386 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.