Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 105 bp
Wild Type = 111 bp
>chr15:101996856-101996966 111bp GAGTCTTCTGATGTCGTGTCCA TCCCTGCACTCTGCCATAC
Wt Sequence:GGTTCTGCCGGGGGATCCAACACTTTCAGCCGCACCACCAAGGCTGTGGTTGTGAAGAAGATTGAAACCCGAGATGGGAAGCTGGTGTCCGAGTCTTCTGATGTCGTGTCCAAGtgaATGGCCACTGAAGTCCTTGCCAGCCTGAGCTCCTGCAGCTGCTCAGGGCTCAcGGGGAGACAGCTGTATGGCAGAGTGCAGGGAACTAGAGACCCGTCCGAGGATCAGCCCTAATCCTCTGGCCAACCTTAGGAGGAATTTCCTATCTGGGATATCCCAATGGCTTCTGCCTCCCA
Mutant Sequence: GCCCTGGCTTCAGCTACGGAATGAGCTCCTTCCAGCCCGGCTTCGGTTCTGCCGGGGGATCCAACACTTTCAGCCGCACCACCAAGGCTGTGGTTGTGAAGAAGATTGAAACCCGAGATGGGAAGCTGGTGTCCGAGTCTTCTGATGTCGTGTCCAAGggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacCTatggtgcccaagaagaagaggaaagtctccaacctgctgactgtgcaccaaaacctgcctgccctccctgtggatgccacctctgatgaagtcaggaagaacctgatggacatgttcagggacaggcaggccttctctgaacacacctggaagatgctcctgtctgtgtgcagatcctgggctgcctggtgcaagctgaacaacaggaaatggttccctgctgaacctgaggatgtgagggactacctcctgtacctgcaagccagaggcctggctgtgaagaccatccaacagcacctgggccagctcaacatgctgcacaggagatctggcctgcctcgcccttctgactccaatgctgtgtccctggtgatgaggagaatcagaaaggagaatgtggatgctggggagagagccaagcaggccctggcctttgaac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49368 | GAG TCT TCT GAT GTC GTG TCC A | Wild type Forward | A | |||
49369 | TCC CTG CAC TCT GCC ATA C | Wild type Reverse | A | |||
49372 | Fluorophore-1 | CTC AGG GCT CAC GGG GAG AC | Quencher-1 | WT Probe | ||
50732 | TTC AGT GGC CAT TCA TTA CTT G | Mutant Reverse | A | |||
50733 | Fluorophore-2 | CTC GGC ATG GAC GAG CT | Quencher-2 | |||
oIMR9102 | ACA TGG TCC TGC TGG AGT TC | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49368 | 0.40 uM |
49369 | 0.40 uM |
50732 | 0.40 uM |
oIMR9102 | 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 |