Protocol 22184: QPCR Assay - Generic Hygromycin
Version 7.0

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

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13585 Fluorophore-1 AAG ACC TGC CTG AAA CCG AAC TGC C Quencher-1 Tg Probe
oIMR1219 CGA GAG CCT GAC CTA TTG CAT Transgene A
oIMR1220 CGA CCG GCT GCA GAA CA Transgene A
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
oIMR1219 0.40 uM
oIMR1220 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
024839 B6.129P2-Pzptm1Vln Mug1tm2Vln/J
034711 B6.Cg-Apptm1.1Dnli/J
035404 B6;129S-Igs7tm166(tetO-EGFP*,CAG-tTA2)Hze/XwyJ
034797 B6;129S4-Gt(ROSA)26Sortm2(CAG-rtTA3,-mKate2)Slowe Col1a1tm1(tetO-GFP,-PRKAA1*)Rshaw/J
034422 B6;129S6-Slc17a7em1(flpo)Hze/J
014594 C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J
003127 FVB;129P2-Bcl3tm1Ver/J
7 strains use this protocol