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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
Mutant= 139 bp
Wild Type = 173 bp
Sequence
Wt Sequence: cccagagctgagcatcctaagcaagcactctgcgaactgagctacatcccagagttcataccaggatttaaggatctcaataggatagaatcaaaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctaGAtaatagcatcttgccttcaaaaacttaactctgactatagagaacaaagacatcttagattcttaattcatgtg
Mutant Sequence:aaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctagTGCGGCCGCACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCCGATCATATTCAATAACCCTTAATataacttcgtataatgtatgctatacgaagttatTAGGTCCCTCGAAGAGGTTCACTAGataatagcatcttgccttcaaaaacttaactctgactatagagaaca
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 35320 | GTT AAG TTT TTG AAG GCA AGA TGC | Common | A | |||
| 35329 | Fluorophore-1 | CGG AAG TCT TGC TTC TAG ATA ATA GC | Quencher-1 | WT Probe | ||
| 36015 | CTG AGC ATC CTA AGC AAG CA | Wild type Forward | A | |||
| 36016 | CGA GGC CCT TTC GTC TTC | Mutant Forward | A | |||
| 36017 | Fluorophore-2 | AGG TCC CTC GAA GAG GTT CAC TAG ATA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 35320 | 0.40 uM |
| 36015 | 0.40 uM |
| 36016 | 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 |
|---|---|
| 033964 | B6.129(Cg)-Ptf1atm2(cre/ESR1)Cvw Krastm4Tyj Ptentm1Hwu/J |
| 006440 | B6.129S4-Ptentm1Hwu/J |
| 013590 | B6.Cg-Tg(Tyr-cre/ERT2)13Bos Braftm1Mmcm Ptentm1Hwu/BosJ |
| 033760 | STOCK Braftm1Mmcm Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033754 | STOCK Brca2tm1Brn Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033752 | STOCK Gt(ROSA)26Sortm1(TMPRSS2/ERG)Key Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033763 | STOCK Gt(ROSA)26Sortm2(myc*T58A)Rcse Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033753 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Brca1tm2Cxd Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033761 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Krastm4Tyj Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033757 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu Tg(ARR2/Pbsn-MYC)7Key/AbshnJ |
| 033751 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033759 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Smad4tm2.1Cxd Ptentm1Hwu/AbshnJ |
| 033755 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 033756 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn/Trp53tm3Tyj Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
| 14 strains use this protocol | |