Protocol 22178: QPCR Assay - iCre QPCR
Version 4.0

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
17817 ACA ACT ACC TGT TCT GCC G Forward A
17818 GCC TCA AAG ATC CCT TCC AG Reverse A
17819 Fluorophore-1 TTC TGC CAC CTC CCA ACT GTC C Quencher-1 Tg Probe
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
ddH2O
Kapa Probe Fast QPCR 1.00 X
17817 0.40 uM
17818 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
010696 B6(129S4)-Et(icre/ERT2)10596Rdav/J
010697 B6(129S4)-Et(icre/ERT2)10727Rdav/J
023691 B6.Cg-Hprttm346a(Ple264-icre/ERT2)Ems/Mmjax
026516 B6.Cg-Tg(Avil-icre/ERT2)AJwo/J
030663 B6;129S-Gcgtm1.1(icre)Gkg/J
016583 C57BL/6-Tg(Slc6a3-icre/ERT2)2Gloss/J
030676 C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J
019098 FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J
021024 FVB-Tg(Csf1r-icre)1Jwp/J
031786 STOCK Prokr2em1(icre)Cfe/Mmjax
032027 STOCK Tg(Avil-icre/ERT2)AJwo/J
030061 STOCK Tg(Tbx18-icre)3Fech/J
12 strains use this protocol