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Stock No: 005734
Protocol 22104: Probe Assay - Generic tTA/rtTA
Version 7.0
Notes
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
Transgene = 79 bp
Internal positive control = 74 bp
This assay can be used for tTA & rtTA but not rtTA Advanced(rtTA*M2)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 13734 | Fluorophore-1 | CGC ATG CCG ACG CGC TAG | Quencher-1 | Tg Probe | ||
| 9342 | GGA CGA GCT CCA CTT AGA CG | Forward | A | |||
| 9343 | CAA CAT GTC CAG ATC GAA ATC | Reverse | A | |||
| 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 |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 9342 | 0.40 uM |
| 9343 | 0.40 uM |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
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 |
|---|---|
| 016198 | 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ |
| 027783 | B6(Cg)-Tg(tetO/Prnp-GCaMP3)600Cai/J |
| 029476 | B6.Cg-Col1a1tm1(tetO-cas9)Sho/J |
| 026262 | B6.Cg-Igs7tm92.1(tetO-ECFP*/Venus*)Hze/J |
| 003563 | B6.Cg-Tg(Cebpb-tTA)5Bjd/J |
| 003767 | B6.Cg-Tg(Eno2tTA)5021Nes/J |
| 003763 | B6.Cg-Tg(Eno2tTA)5030Nes/J |
| 005964 | B6.Cg-Tg(GFAP-tTA)110Pop/J |
| 002618 | B6.Cg-Tg(MMTVtTA)1Mam/J |
| 006232 | B6.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
| 028295 | B6.Cg-Tg(tetO-DRD3)3-10Kndl/J |
| 034533 | B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ |
| 012433 | B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J |
| 007678 | B6;SJL-Tg(KRT14-rtTA)208Jek/J |
| 006242 | C.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
| 006245 | C.Cg-Tg(SFTPC-rtTA)5Jaw/J |
| 013585 | FVB-Tg(Cdh5-tTA)D5Lbjn/J |
| 005625 | FVB-Tg(Pcp2-tTA)3Horr/J |
| 006222 | FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
| 006225 | FVB.Cg-Tg(SFTPC-rtTA)5Jaw/J |
| 006209 | FVB.Cg-Tg(Tal1-tTA)19Dgt/J |
| 034802 | FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax |
| 004127 | FVB/N-Tg(Nes-rtTA)306Rvs/J |
| 005942 | FVB/N-Tg(Pf4-tTA/VP16)42Kra/J |
| 006875 | FVB/N-Tg(Tagln-rtTA)E1Jwst/J |
| 004937 | NOD.Cg-Tg(Ins2-tTA)1Doi/DoiJ |
| 005734 | NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ |
| 006999 | STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J |
| 018999 | STOCK Gt(ROSA)26Sortm1(tTA,tetO-Mir155)Fjsl/J |
| 024108 | STOCK Igs7tm93.1(tetO-GCaMP6f)Hze Tg(Camk2a-tTA)1Mmay/J |
| 024854 | STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J |
| 003272 | STOCK Tg(Cebpb-tTA)5Bjd/J |
| 003273 | STOCK Tg(CMV-rtTA)4Bjd/J |
| 003271 | STOCK Tg(CMV-tTA)3Bjd/J |
| 005493 | STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J |
| 008790 | STOCK Tg(tetO-DISC1*)1001Plet/J |
| 36 strains use this protocol | |