For in-depth product & services help, ask our
Technical Information Scientists
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.
Archived Notes:
Name of protoocl changed from Tg(Il2/NFAT-luc)83Rinc to generic Luc qPCR 12-13-17 ESP
Removed 13745 since it had a Fam label and replaced with 105 12-18-17 ESP
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 |
|---|---|---|---|---|---|---|
| 13736 | Fluorophore-1 | TGG GCG CGT TAT TTA TCG GAG TTG 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 | |||
| oIMR6615 | CTC TTC AAT TCT TTA TGC CGG TG | Transgene Forward | A | |||
| oIMR6616 | TTC GCG GGC GCA AC | Transgene 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 |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| oIMR6615 | 0.40 uM |
| oIMR6616 | 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 |
|---|---|
| 039259 | B6.Cg-Gt(ROSA)26Sortm1.2(CAG-rtTA3,-GFP*,-luc*)Zhrt Tyrc-2J/ZhrtJ |
| 033605 | B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J |
| 006098 | B6.Cg-Tg(Il2/NFAT-luc)83Rinc/J |
| 006851 | B6.Cg-Tg(Per1-luc)025Jt/J |
| 006101 | B6.Cg-Tg(TRE/Prl-luc)31FlvRinc/J |
| 027662 | C57BL/6-Tyrc-Brd Tg(Gh1-luc/EGFP)D8Mrln/J |
| 039260 | C57BL/6J-Bdnfem1Bxu/J |
| 007800 | FVB/N-Tg(Ins1-luc)VUPwrs/J |
| 012370 | FVB/NJ-Tg(Hspa1a-luc,-EGFP)2Chco/J |
| 017597 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#bAhmb/J |
| 10 strains use this protocol | |