Stock No: 029231
Protocol 30399: Probe Assay - Bhlha15<tm4Skz>-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= 91 bp

Wt= 83 bp

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34518 CAC CAC CTC CTG GGA AAA C Common A
34519 CTG TGT CCT GCA TGG GTG T Wild type Reverse A
34520 GCT CGA GAT AAC TTC GTA TAG CA Mutant Reverse A
34521 Fluorophore-1 AGC CTG GAG CTA TGA AGA CCA Quencher-1 WT Probe
34522 Fluorophore-2 CCG CAT AAC TTC GTA TAA TGT ATG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34518 0.40 uM
34519 0.40 uM
34520 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.