Protocol 21449: Probe Assay - Arl2bp<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= 111 bp

Wt= 119 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32479 TGA TTG TCA TAC CGG TTT TCG Mutant Forward A
32480 Fluorophore-1 CAC GAG TGT TTT CCT GTG TTT G Quencher-1 MUT Probe
32481 TCC TAT GGT CAA TTC CTT GAT T Wild type Forward A
32482 Fluorophore-2 AAT CAA GTA GAC CCT TGC TTT ACA A Quencher-2 WT Probe
32483 AGC CAT AGT CTC CTG GCA AC Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32479 0.40 uM
32481 0.40 uM
32483 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.