Protocol 30484: Probe Assay - Rnf24<em1J> Probe -Alternate 6
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

Mut= 182 bp

Wt= 181 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34189 TAG GCT GAG ACA TCA AGC ACA C Wild type Forward A
34190 CTG TGG TAT AAA CTG ACA AAG G Wild type Reverse A
34192 ACA GTG GGA TAT GCA CTC CA Mutant Reverse A
34193 Fluorophore-1 CTT TGA GAC TGA GTT CCA TTC ATA GT Quencher-1 WT Probe
34194 Fluorophore-2 CTG TAA CAC TTC AAG TCA CCA CTG Quencher-2 MUT Probe
36002 ACA AAT TAA CTA TTG AAC TAA GAC TTT GC Mutant Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34189 0.40 uM
34190 0.40 uM
34192 0.40 uM
36002 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.