Protocol 31472: QPCR Assay - Tg(IL15)1Sz
Version 1.0

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=   72 bp

IPC = 74 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36944 GGT GGC CTG ACC TAA GGA AG Transgene Forward A
36945 GTG CCA TCG AGA CTT CCA CT Transgene Reverse A
36946 Fluorophore-1 TGC CAT AGC AGG TAA TAC CAG CTT 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
36944 0.40 uM
36945 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
037322 NOD.Cg-Flt3em2Mvw Prkdcscid Il2rgtm1Wjl Tg(FLT3LG)7Sz Tg(IL15)1Sz/J
037320 NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav Tg(IL15)1Sz/J
030890 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ
3 strains use this protocol