Genotyping Protocols Database
Stock No: 003658
Protocol 30028: QPCR Assay - humanized GFP/ER fusion qPCR
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 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 = 67 bp

IPC = 74 bp

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
oIMR1858 TGG TCC CAA TTC TCG TGG AA Transgene A
oIMR1859 CCT CTC CGC TGA CAG AAA ATT T Transgene A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0031 Fluorophore-1 TGC CCA TTC ACA TCG CCA TCC A Quencher-1 Tg Probe
TmoIMR0105 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
oIMR1858 0.40 uM
oIMR1859 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
003658 STOCK Tg(TIE2GFP)287Sato/J
004659 B6.Cg-Tg(TIE2GFP)287Sato/1J
006207 STOCK Tg(Pcp2-cre)1Amc/J
006770 STOCK Rag1tm1Mom Tg(TIE2GFP)287Sato/J
4 strains use this protocol