Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 113 bp
Wild Type = 129 bp
>chr7:105293225-105293353 129bp CTAGCTGCTCCTCCTGTGCT CAGCATCCGGCTTCTAGACT
Wt Sequence: GACCTCACCAACCTCATGACACGCCAGCTGCTGGACCCCGCCGCCATCTTCTGGCGCAAGGAAGACTCCGACGCCATGGATTGGAATGAGGCAGACGCCCTGGAGTTTGGGGAGCGCCTTTCTGATCTGGCCAAGATCCGCAAGGTCATGTATTTCCTCATCACCTTTGGCGAGGGCGTGGAGCCTGCCAACCTAAAGGCCTCTGTGGTGTTTAACCAGCTCTGATGACAGCCCTGGCTGCCCTACCCCTGGCCCCACCTCTCCCTTGCCTGGATCTCCTTCCTCATGTGTATTTTGGGGACATTCTTCTAGCTGCTCCTCCTGTGCTCATCTTGGCCAGAGTTCCCCCGAGTGCTACATCCCCTCCTTTTCCCTGGTGCCAGTGCTGCGGCTCACAGTGATGTCCCATGGCTCCGTAGTCTAGAAGCCGGATGCTGCTACTATAGACTGTAGAGGCCTTTTGGGTCCACGTGGGAAGATGGATGGGCCCCCTGTGGTGAAGAGCGGGACTGAGAGATAAAGAGACTGACCAAGAGATGCAAACGGCCAG
Mutant Sequence: AGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAGAATTCGCTAgcttcaaaagcgctctgaagttcctatactttctagagaataggaacttcggaataggaacttcaagatccccgggtaccACGCGTTAATGGCTAATCTCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38925 | CGG TAG AAT TCG CTA GCT TCA | Mutant Forward | A | |||
38926 | GAT TAG CCA TTA ACG CGT GGT | Mutant Reverse | A | |||
38927 | CTA GCT GCT CCT CCT GTG CT | Wild type Forward | A | |||
38928 | CAG CAT CCG GCT TCT AGA CT | Wild type Reverse | A | |||
38929 | Fluorophore-1 | ACT TCG GAA TAG GAA CTT CAA GAT CCC | Quencher-1 | MUT Probe | ||
38930 | Fluorophore-2 | ACA TCC CCT CCT TTT CCC TGG T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38925 | 0.40 uM |
38926 | 0.40 uM |
38927 | 0.40 uM |
38928 | 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 |