Stock No: 002244
Protocol 38216: QPCR Assay - Tg(Apoa2)1Lus
Version 2.0

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

Tg=   84 bp

IPC = 74 bp

This assay was not tested on homozygotes so it may or may not be able to distinguish hemi from hom

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52551 CCT GCG GCT AAG TGA GAT GT Transgene Forward A Apoa2
52552 AGG TAG GGA GAG GGG CTC TA Transgene Reverse A Apoa2
52553 Fluorophore-1 CCA GGC CCA GTC TTC CCA C Quencher-1 Tg Probe
oIMR1544 CAC GTG GGC TCC AGC ATT A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
52551 0.40 uM
52552 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

This is the only strain that uses this protocol.