Protocol 30045: QPCR Assay - Tg(HBA-HBBs)41Paz
Version 7.0

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

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
oIMR1799 GGA ATG GAA AGG AAA GTG AAT ATT TG Transgene A
oIMR1800 GCT GTC TCC TAG CAA CGA CTT CT Transgene A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0027 Fluorophore-1 CCC ACC TCC AGT GTA ACT GCC TAG TCT TTC ATA 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
oIMR1799 0.40 uM
oIMR1800 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
003342 STOCK Hbatm1Paz Hbbtm1Tow Tg(HBA-HBBs)41Paz/J
021452 STOCK Hbbd3th Tg(LCR-HBA1,LCR-HBB*)1Tow/J
2 strains use this protocol