Protocol 38170: Probe Assay - Tg(K18-ACE2)2Prlmn QPCR
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= 88 bp

IPC= 74 bp

Sequence

Tg Sequence:

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52079 GGA TGG AGT ACC GAC TGG AG Transgene Forward A hACE2 cDNA
52080 CAT ATG CTT TAT CTC CAA GAG CTG Transgene Reverse A hACE2 cDNA
52081 Fluorophore-1 AGA CCA AAG CAT CAA AGT GAG G Quencher-1 Tg Probe hACE2 cDNA
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 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52079 0.40 uM
52080 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
035077 B6.Cg-Stat1tm1Dlv Tg(K18-ACE2)2Prlmn/J
034902 B6.Cg-Tg(FCGRT)32Dcr Tg(K18-ACE2)2Prlmn Fcgrttm1Dcr/J
035041 B6.Cg-Tg(K18-ACE2)2Prlmn Ifnar1tm1.2Ees/J
034860 B6.Cg-Tg(K18-ACE2)2Prlmn/J
035247 C.Cg-Tg(K18-ACE2)2Prlmn/J
034901 NOD.Cg-Tg(K18-ACE2)2Prlmn Prkdcscid Il2rgtm1Wjl/J
6 strains use this protocol