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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= 80 bp
IPC = 74 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76753 | GCA GAA CAA GTA CCT GGG | Transgene Forward | A | FLPo | ||
| 76754 | AAA AGT AGA TGT GCC TGG AC | Transgene Reverse | A | FLPo | ||
| 76755 | Fluorophore-1 | TGG TGA CCG AGA CCA AGA CA | Quencher-1 | Tg Probe | FLPo | |
| oIMR1544 | CAC GTG GGC TCC AGC ATT | A | ||||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | 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 |
| 76753 | 0.40 uM |
| 76754 | 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 |
|---|---|
| 040970 | B6(Cg)-Brs3em1(flpo)Rei/J |
| 038534 | B6(Cg)-Foxp3tm1.1(flpo)Daav/J |
| 012930 | B6.129S4-Gt(ROSA)26Sortm2(FLP*)Sor/J |
| 039198 | B6.129S6-Ptf1atm1(flpo)Hcrd/J |
| 033952 | B6.129S7(FVB)-Dbhtm1.1(flpo)Pjen/J |
| 035532 | B6.Cg-Sntb1tm1.1Scf/J |
| 039739 | B6.Cg-Ucmatm1(flpo)Hze/J |
| 011065 | B6.Cg-Tg(Pgk1-flpo)10Sykr/J |
| 039199 | B6.Cg-Tg(Sox2-flpo)4.2.28Mhem/J |
| 033850 | B6;SJL-Tg(Chat-flpo)22Majh/J |
| 040187 | C57BL/6-Adora2aem2(flpo)Rcos/J |
| 040716 | C57BL/6-Avptm1.1(flpo)Gdol/J |
| 040189 | C57BL/6-Drd1em2(flpo)Rcos/J |
| 040184 | C57BL/6-Slc6a3em1(flpo)Rcos/J |
| 041282 | C57BL/6-Trpa1tm1.1(flp)Ttc/J |
| 041283 | C57BL/6-Trpv1tm1.1(flp)Ttc/J |
| 040836 | C57BL/6J-Cux1em1(flpo)Zjh/J |
| 040838 | C57BL/6J-Igfn1em1(flpo)Zjh/J |
| 041616 | C57BL/6J-Tac1em1(flp)Pc/J |
| 040837 | C57BL/6J-Wfs1em1(flpo)Zjh/J |
| 030288 | FVB/N-Tg(Cdx2-flpo)2Ddg/J |
| 039436 | FVB/N-Tg(MMTV-flpo)9Gcr/J |
| 038629 | STOCK Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze Pdgfrbtm1.1(flpo)Leol/LeolJ |
| 030755 | STOCK Htr3atm1.1(flpo)Rudy/J |
| 040326 | STOCK Mettl3tm1.1Jcbr/MgkJ |
| 038627 | STOCK Pdgfratm1(flpo)Leol Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/LeolJ |
| 039795 | STOCK Pthlhtm1(flpo)Zjh/J |
| 039724 | STOCK Vimtm2(flpo)Ggen/J |
| 28 strains use this protocol | |