Stock No: 005703
Protocol 20843: QPCR Assay - Generic FLPe 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=   145 bp

IPC = 74 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25140 GCT GAA CTA ACC TAT TTA TGT TGG ATG Transgene Forward A
25141 GCG TCT TGT ATT TAA ACT GGA GTG Transgene Reverse A
25142 Fluorophore-1 TGG CTC TCT TGA TTG CTG TTC CGT Quencher-1
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

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
25140 0.40 uM
25141 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
005703 B6.Cg-Tg(ACTFLPe)9205Dym/J
003800 B6;SJL-Tg(ACTFLPe)9205Dym/J
034712 B6J.Cg-Cyp27a1tm1.1Dpm/J
034731 B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J
019100 B6N.Cg-Tg(ACTFLPe)9205Dym/CjDswJ
035185 STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ
6 strains use this protocol