Stock No: 008320
Protocol 31455: Probe Assay - Lepr<tm2(cre)Rck> Probe-Alternate 2
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= 100 bp

Wt= 149 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36248 CTC TAG AAA GTA TAG GAA CTT CAG AGC Mutant Forward A
36251 Fluorophore-1 TGA AGC TGG GTA CAT TAA TTA AGC GG Quencher-1 MUT Probe
36913 GGA CCT CTG GTG AGA ACT TTG TAC Wild type Forward A
36914 TGA CAG GCT CTA CTG GAA TGG Common A
36915 Fluorophore-2 ACT TAA CTG TGT AAT CTC ATC CAA G Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36248 0.40 uM
36913 0.40 uM
36914 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.