Protocol 21448: Probe Assay - Tbc1d16<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

Mut= 113 bp

Wt= 101 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32474 GAA CGA CAA GCC AAG AAA GC Common A
32475 CAG ATG GGA GAC CCA ATC AT Wild type Reverse A
32476 Fluorophore-1 TGA CTG GAG GAG ACC CAA GT Quencher-1 WT Probe
32477 Fluorophore-2 CAC AGG ATC TTT GGA CTT TTC TG Quencher-2 MUT Probe
32478 CTG AGA CAC ACC TGA CAC TCT TC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32474 0.40 uM
32475 0.40 uM
32478 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.