Genotyping Protocols Database
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 4.0 -- 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
002618 B6.Cg-Tg(MMTVtTA)1Mam/J
003271 STOCK Tg(CMV-tTA)3Bjd/J
003272 STOCK Tg(Cebpb-tTA)5Bjd/J
003273 STOCK Tg(CMV-rtTA)4Bjd/J
003563 B6.Cg-Tg(Cebpb-tTA)5Bjd/J
003763 B6.Cg-Tg(Eno2tTA)5030Nes/J
003767 B6.Cg-Tg(Eno2tTA)5021Nes/J
004127 FVB/N-Tg(Nes-rtTA)306Rvs/J
004937 NOD.Cg-Tg(Ins2-tTA)1Doi/DoiJ
005493 STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J
005625 FVB-Tg(Pcp2-tTA)3Horr/J
005734 NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ
005942 FVB/N-Tg(Pf4-tTA/VP16)42Kra/J
005964 B6.Cg-Tg(GFAP-tTA)110Pop/J
006209 FVB.Cg-Tg(Tal1-tTA)19Dgt/J
006222 FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006225 FVB.Cg-Tg(SFTPC-rtTA)5Jaw/J
006232 B6.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006235 B6.Cg-Tg(SFTPC-rtTA)5Jaw/J
006242 C.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006245 C.Cg-Tg(SFTPC-rtTA)5Jaw/J
006875 FVB/N-Tg(Tagln-rtTA)E1Jwst/J
006999 STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
007678 B6;SJL-Tg(KRT14-rtTA)208Jek/J
008250 STOCK Tg(Ins2-rtTA)2Efr/J
008790 STOCK Tg(tetO-DISC1*)1001Plet/J
013585 FVB-Tg(Cdh5-tTA)D5Lbjn/J
016198 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ
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
026262 B6.Cg-Igs7tm92.1(tetO-ECFP*/Venus*)Hze/J
027783 B6(Cg)-Tg(tetO/Prnp-GCaMP3)600Cai/J
028295 B6.Cg-Tg(tetO-DRD3)3-10Kndl/J
029476 B6.Cg-Col1a1tm1(tetO-cas9)Sho/J
032106 B6.Cg-Tg(Myh6-tTA)6Smbf Tg(tetO/CMV-ATP2B4,-lacZ)1Husa/Mmjax
032712 STOCK Col1a1tm1.1(tetO-Gfi1,-Venus)Hlg/J
37 strains use this protocol