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Stock No: 035560
Protocol 38260: QPCR Assay - Generic tdTomato QPCR
Version 2.0
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
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 |
| 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 |
|---|---|
| 035560 | B6(129S4)-Tg(tetO-Cxcl2,-tdTomato)#Forsb/Mmjax |
| 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 |
| 034311 | NOD/ShiLtJ-Cd8aem1Doi/J |
| 038629 | STOCK Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze Pdgfrbtm1.1(flpo)Leol/LeolJ |
| 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 |
| 8 strains use this protocol | |