Stock No: 005852
Protocol 30263: QPCR Assay - Phex<Hyp-Duk>-Alternate 1
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

Mouse wt =   80 bp

IPC = 74 bp

Plese note this is a qPCR for mouse Exon 14 on the X chromosome so Female mouse wt will have two copies (hom-like); Female hets and wt Males will have one copy (tg/o-like) and Female homs and Male hemis will have no copies (wt-like)

X-linked

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33653 Fluorophore-1 CAA CGT ACT GCA AAC CCG CAA GTA TT Quencher-1
34225 GAT CAA ATT TTC AGA ATC GGA CTA C Transgene Forward A
34226 GCC AGA AGA AAT CAG ACT GTG C Transgene Reverse A
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34225 0.40 uM
34226 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
005068 B6.C-PhexHyp-Duk/J
005852 BALB/c-PhexHyp-Duk/J
003905 BALB/cAnBomUrd-PhexHyp-Duk/J
3 strains use this protocol