Protocol 21452: Probe Assay - Xkr4<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= 103 bp

Wt= 113 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32507 AAG GCA CTT CCC TTT GAG CTA T Mutant Forward A
32508 Fluorophore-1 TGA CCA AAT TCC TTG TTC TGT G Quencher-1 MUT Probe
32509 TCT GGA AAG TGC TCC ACA AT Wild type Forward A
32510 Fluorophore-2 TAC AGG CCC TCC AAG GTA AG Quencher-2 WT Probe
32511 TCT CTC TCC CAA TTC AGA AAC C Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32507 0.40 uM
32509 0.40 uM
32511 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.