Genotyping Protocols Database
Protocol 20498: QPCR Assay - Generic APP human genomic or cDNA
Version 5.0

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. Use an appropriate instrument-specific Fuorophore/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

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
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
oIMR5260 TGG GTT CAA ACA AAG GTG CAA Transgene Forward A
oIMR7707 GAT GAC GAT CAC TGT CGC TAT GAC Transgene Reverse A
TmoIMR0076 Fluorophore-1 CAT TGG ACT CAT GGT GGG CGG TG Quencher-1 Tg Probe
TmoIMR0107 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
oIMR1544 0.40 uM
oIMR3580 0.40 uM
oIMR5260 0.40 uM
oIMR7707 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
004661 B6.Cg-Zbtb20Tg(PDGFB-APPSwInd)20Lms/1J
004662 B6.Cg-Tg(PDGFB-APP)5Lms/J
005300 B6.129-Tg(APPSw)40Btla/Mmjax
005301 B6.129S2-Tg(APP)8.9Btla/J
006406 B6.129S4-Tg(APPSwLon)96Btla/Mmjax
006409 129S1.129(Cg)-Tg(APPSw)40Btla/Mmjax
006472 D2.129(B6)-Tg(APPSw)40Btla/Mmjax
006555 A.129(B6)-Tg(APPSw)40Btla/Mmjax
007027 C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax
008730 B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax
031988 129S4.Cg-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/LfaJ
11 strains use this protocol