Protocol 29948: Probe Assay - Zfp7<em1J> Probe Alt 1
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.

 

3' end has lots of gt repeats - had to make large products and mut probe is across a string of 5g's which im not thrilled about.

 

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=  96 bp

Wild Type =138 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31973 CCA CAT CCT ACC CTT TCC AC Common A
31975 Fluorophore-1 CCC TGA GGG GGT GAA TAA AT Quencher-1 WT Probe
31980 CTC CCC ACT GAA CAG ACA CA Wild type Reverse A
33697 Fluorophore-2 ACT GTC AGA GAC GGG GGT G Quencher-2 MUT Probe
33698 CCA AGC AGT TAG CAC CAC AC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31973 0.40 uM
31980 0.40 uM
33698 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.