Protocol 37809: Probe Assay - Generic tdTomato Probe Alternate1
Version 1.0

Notes

This assay cannot distinguish hemi from hom

 

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=  63 bp

IPC = 74 bp

Sequence

Tg Sequence: ACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGcaggacggcacgctgatcTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52208 GTG ACC GTG ACC CAG GAC Transgene Forward A
52209 CGC GCA TCT TCA CCT TGT Transgene Reverse A
52210 Fluorophore-1 CAG GAC GGC ACG CTG ATC Quencher-1 Tg Probe tdTomato
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
52208 0.40 uM
52209 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
035560 B6(129S4)-Tg(tetO-Cxcl2,-tdTomato)#Forsb/Mmjax
030219 B6.Cg-Igs7tm139.1(tetO-EGFP,CAG-tdTomato,-tTA2)Hze/J
037382 B6.Cg-Igs7tm224(CAG-EGFP,CAG-dTomato)Tasic/J
037383 B6.Cg-Igs7tm227(CAG-EGFP,CAG-tdTomato)Tasic/J
033125 B6.FVB-Tg(tetO-Bcl2,-tdTomato)LM1Ryu/J
036087 B6;129-Gt(ROSA)26Sortm3Cos/J
018936 B6;C-Tg(Erv4-tdTomato)449Slp/J
036272 B6;CBA-Gt(ROSA)26Sorem1(CAG-Ikzf2,-tdTomato)Zyliu/J
027395 C57BL/6-Tg(Pvalb-tdTomato)15Gfng/J
038819 STOCK Gt(ROSA)26Sorem3(CAG-Insm1,-tdTomato)Zyliu/J
038629 STOCK Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze Pdgfrbtm1.1(flpo)Leol/LeolJ
030292 STOCK Ntrk3tm2.1Ddg/J
038627 STOCK Pdgfratm1(flpo)Leol Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/LeolJ
038628 STOCK Pdgfratm2.1(flpo)Leol Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/LeolJ
026690 STOCK Tg(Ucp1-luc2,-tdTomato)1Kajim/J
15 strains use this protocol