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= 134 bp
Wild Type = 152 bp
Wt Sequence:aagaggggaggagctacttgcatAGTTGttcaattttaatttggcagtagggtattgaaagacctttcatattttattcaacaattttctttccctatttaagGTATATCTTCCAATCACAGTGATGATTATCCAtaTAGCTGGTCTTTTCCACCTTATCATCCATCCTCAGAGAAATATGAAAACACTAAGgtaggtctgtacaagg
Mutant Sequence: AACCCCAAAGAGGGGAGGAGCTACTTGCATAGTTGTTCAATTTTAATTTGGCAGTAGGGTATTGAAAGACCTTTCATATTTTATTCAACAATTTTCTTTCCCTATTTAAGGTATATCTTCCAATCACAGTGATGATTATCCATcgggatccgcggtctagacttctcttcccacacgCAGTCCTCTTTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35691 | TGG CAG TAG GGT ATT GAA AGA | Common | A | |||
35692 | GAC TGC GTG TGG GAA GAG A | Mutant Reverse | A | |||
35693 | CCT TAG TGT TTT CAT ATT TCT CTG AGG | Wild type Reverse | A | |||
35694 | Fluorophore-1 | ATC CAT ATA GCT GGT CTT TTC CAC CT | Quencher-1 | WT Probe | ||
35695 | Fluorophore-2 | CGG GAT CCG CGG TCT AGA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35691 | 0.40 uM |
35692 | 0.40 uM |
35693 | 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 |