Protocol 30597: Probe Assay - Zscan5b<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= 82 bp

Wt= 104 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34969 GGG ATA ATA GGG TGG GCT CT Common A
34970 GCA AAG GAA TAA TAA AAA GAC CAT T Mutant Reverse A
34971 Fluorophore-1 TTC CCA CAA GTC TGG TAT GG Quencher-1 MUT Probe
34972 Fluorophore-2 TTC CCA CAA CTC TGG AAA AA Quencher-2 WT Probe
34973 ACG GAG AGA TGG AGC ACA GA Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34969 0.40 uM
34970 0.40 uM
34973 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.