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.
Mutant= 139 bp
Wild Type = 173 bp
Wt Sequence: cccagagctgagcatcctaagcaagcactctgcgaactgagctacatcccagagttcataccaggatttaaggatctcaataggatagaatcaaaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctaGAtaatagcatcttgccttcaaaaacttaactctgactatagagaacaaagacatcttagattcttaattcatgtg
Mutant Sequence:aaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctagTGCGGCCGCACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCCGATCATATTCAATAACCCTTAATataacttcgtataatgtatgctatacgaagttatTAGGTCCCTCGAAGAGGTTCACTAGataatagcatcttgccttcaaaaacttaactctgactatagagaaca
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 |
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 |
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 |
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 |