Protocol 20486: Probe Assay - Foxn1<em1j>del2-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=  89 bp

Wild Type = 91 bp

Sequence

Wt Sequence: ATCCCTATCAGAGGATTGCACCCCAGGCcAAcgCCGAAGGTCACCAGCCAC

 

Mutant Sequence:ATCCCTATCAGAGGATTGCACCCCAGGC(-)AAtCCGAAGGTCACCAGCCAC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
30546 CTA CCT GGG CTC ACC TCA CT Forward A
30547 GTA GAT GGG CTT TGG GAA GA Reverse A
30548 Fluorophore-1 CAG GCC AAC GCC GAA GGT Quencher-1 WT Probe
30555 Fluorophore-2 CAG GCA ATC CGA AGG TCA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
30546 0.40 uM
30547 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 4.0 -- 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.