Protocol 21282: Probe Assay - Myo5c<em1J>Probe Alt 1
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= 144 bp

Wt= 140 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31201 Fluorophore-1 CAA GCA AAC CAT CCA CAC AC Quencher-1 WT Probe
31202 Fluorophore-2 ATG GGC TCG TGT CTG AGT CT Quencher-2 MUT Probe
32112 CTT GTG GCT GCT GCA GGT Common A
32113 GAC AAC AAT GTC CCA AGA AGT AA Mutant Reverse A
32114 ACA CCA CCA AAA CCC AAA TC Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32112 0.40 uM
32113 0.40 uM
32114 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.