Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 127 bp
Wild Type = 132 bp
>chr9:34996667-34996798 132bp AGCCACACAGGATGGTCTCT CTGTCCACGTGTGTTGGTG
Wt Sequence:GCTATGCCAGGCACTGAGTCGTAGTCACTGCCGTGTGCTGCTCATCACTCCAGGCTTCCTTCGGGACCCCTGGTGCAAGTACCAGATGCTGCAGGCCCTGACGGAGGCCCCGGCGTCGGAGGGTTGCACCATACCCCTGCTGTCCGGCCTGTCCAGAGCCGCCTATCCGCCGGAACTCCGATTCATGTACTATGTGGATGGCAGAGGCAAGGACGGAGGCTTTTACCAAGTCAAGGAGGCTGTTATACACTgtaagatatagGGGGGCTGAGAAGGGGGACAGGAGGCATCCAGTGTCtagataagaacttTAGGAGGTAGTCTCTGCAGTGCCAGTGAATGGGAAAGCTCCTGGAGAATACTGGCTGGATTTGTACCCTGGCAT
Mutant Sequence: aaatgtgtcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagttctaattccatcagaagctatAACTTCGTATAATGTATGCTATACGAAGTTATctagagtcGACAGGAGGCATCCAGTGTCTAGATAAGAACTTTAGGAGGTAGTCTCTGCAGTGCCAGTGAATGGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
27228 | TGC CAA GTT CTA ATT CCA TCA G | Mutant Forward | A | |||
39504 | Fluorophore-1 | CTG GCA CTG CAG AGA CTA CCT | Quencher-1 | Mutant Reverse | A | |
39505 | Fluorophore-2 | AGC CAC ACA GGA TGG TCT CT | Quencher-2 | Wild type Forward | A | |
39506 | CTG TCC ACG TGT GTT GGT G | Wild type Reverse | A | |||
39507 | Fluorophore-3 | CGA CAG GAG GCA TCC AGT GTC | Quencher-3 | MUT Probe | ||
39508 | Fluorophore-4 | CAC CCC CGA GTC ACA GTT CAC | Quencher-4 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
27228 | 0.40 uM |
39504 | 0.40 uM |
39505 | 0.40 uM |
39506 | 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 |