Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 117 bp
Wild Type = 116 bp
>chr2:30472246-30472361 116bp GACGTTCCGCTCTCCTGAGT GAGGAAATGTGACCGAAACG
CGCGCACCTGCCGCAGGTGAGGCCCAACCATGCGGAAGGGGGCGGAGCCATCTCCGCTGCGCTCAGGTGAGCCGACCCCCAGCTCGCAGGAGCCACGTCCAGGAAAGCTACGAATTTCGACGTGGCTCCCAAGAGGCGACCCAAAGCTGAGAGAGACTACCTCGGAGGCTCCTAGTCCcacgaggtgccgcggccccaggagaacattcgaggcaacggcggcgttggaggcccggggtccgttcgttatttgaatagcacaaaggaaccgccttggtctgattggccagtcaagtggtcccggggagggcagtggctgtcagtcggaaggctctacacgcttctattggctaatctacagtggggcggggcgctgctgcccagctaaatagttggggccggggccggcggaggctcattgctttggcgccgtctggggagcgtgagcctgcgggtggcgcgcaacgcatggtggcgacttctctcggagtgtcgccgacccgccgacccgggctcggttccctgtgcctggctcgcccgggcgaccaactggagacacacgcaaggagcagcattcgcagcaccagaggcggctgctccgccgcccgcgtctccgcgggagcatggagtgcgccctggacgcccagagcctgatcagcatctccctgcgcaagatccacagctcccggacccagcgcggcggcatcaagctgcacaagaacctcctggtgtcctacgtgctccgcaacgcgcgccagctctacctgagcgagcgttacgccgagctctaccggcgccagcagcagcagcagcaacagcagccgccccaccaccagcaccagcacctcgcttacgcggcgcccggaatgccggccagcgcggccgacttcggcccgctccaacttggcggcggcggggacgcggaggcgcgcgagccggttgcccggcaccagctgcaccagctccaccagcttcaccagctgcacctccagcagcagctgcaccaacaccaacacccggcgcccaggggctgcacggcggcggcgccggtggcggtggccggggcgcccgcgggctgcgcgggggcgctctcggagctgcccgggtgcgctgcgctccagccgccgcacggcgcgccccaccgcgggcagcacttggagccgctgcagccgggtcccgcgccgctgccgccgcccgcgcccgccgcgctctgcccgcgggaccctcgcgtcccggccgcttgctctgcgccctcggtgcttccgggggctgccccttcgaccgtcgctgcctcctctccgcccgcctctacagccccttcctcatccactggcttctaccggggcgcgtacccggccccctcggacttcggcgtgcactgcagcagccagaccaccgtgctggacctggacactcacgtggtgaccacggtagaaaacggctacttgcaccaggactgctgcgcctccgcccactgcccctgctgtggccagggcgctccgggacccggtctggcgtccgccgctggttgcaagcgcaagtattaccctggccaggaggaagaggacgacgaagaggacgcgggcgacctgggagccgagccccccgggggtaccccattcgccccctgtaagcgcgcccgcttcgaggacttctgcccggactcgtccccggacgcgtccaacatctcaaacttgatctccatctttggctcgggcttctcggggctggtgagccgacagccggactcctcggagcagccgccgccgctcaacgggcagctgtgcgccaagcaggcgctcgccagcctcggcgcctggactcgagccattgtcgccttctagggacccccgagggcatggggaccctgggccccgcggggctggggccagacaaagactcggccaaggggcgatagggagcgagcgggtcctggccactcggggctgagctggggacgagcagaggctgatgttttataaattgtaaaataaaaaaaaaaaatctaaaaatcttggactttatttttgcagagaaaaagcgcctatttaagtatgctttgtgtttctcttactcttttccccccctttttattgtactgattgcagtggtgtttagcgaggagcctgccacgtgaggcagggctgctgcccggaggaggtgcccggccgccgggggcgaggctgggtgcccgggcgcgccaggggcgcaacgcaggagggcgcggggccccggacgccgattctaatcagttgtcaagagccgggaagcccgacgttccgctctcctgagtgcctttctggggtgaggaacgggtcttgtgaaaccctgagcaaaaaaacaaaggcaaactctatctccaaaagggacgtttcggtcacatttcctctctgggggcggcctccaaagttctcaaaatgagaaggcagaaatgaaaacacttcaacttttttttcttttcttgccgggttgggcgtctagaacccctcctctccccgcccctctggctccgtcctcctccccctcctccacccgcctcgcggactcgggcgggggggggggggggcaccctgacactctcagcacagttgggaaaggggtggagggcgagcagggcgaactacatttcccatcatgcccagcactgcggtcctcactaaacaaaaaaggaagtcgattccttcacctggatccccgggggagggaggggcccggaccgccgactgagctggagacttttcgcttagctcctggtcagctctagtgattgtgcgcgcttctaagttaccttgtgctttagccttcggtcccatctcatgaaaccagcattgttccagcctgtgagaaccctgtctccgtcctcagcgcgatagattttgtttaatttaaaagccttttgttgtgagaaggtgaggttcgcttagtgcccctctgggtctctgccatcaacaccccgaggactccaaaacaagttgccaaccttgccttcccggcccttcttcctcctgtatttttgtgcataatgaattgtatctcaccgggtaaaactgttcaggttatttgtttaaatttataatcttaataaaaagtcaattgtagagtgtatggtggttcctttgctttcacatgggcttagaggtgagaaaatctaaacacacacacacacacacacacacacgcaagcaaacaccagctttccagggccatggtggaaacccatggatacccccatatcCGCAGGGCCAGGGTTGGAATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCTGGGGATGGAGCTAGGGTTGGGAGTCAGTATGTGTGCGTTCTGGGGATGGAGGCCGGTGCTACAAGGTCTTCTCCATAAGCCCTCCTCACCAGCGGGTCTTGCCCCCTTGGGAACCCGCTGCCCAGCAGGAAGCTGAGCCAGCCTCCAACATATTTCCATG
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44453 | GAC GTT CCG CTC TCC TGA GT | Wild type Forward | A | |||
44454 | GAG GAA ATG TGA CCG AAA CG | Wild type Reverse | A | |||
44455 | Fluorophore-1 | CCT TTC TGG GGT GAG GAA CG | Quencher-1 | WT Probe | ||
44456 | CAC ACA CAC ACA ATT CCA ACC | Mutant Reverse | A | |||
44457 | CAC GTC CAG GAA AGC TAC GA | Mutant Forward | A | |||
44458 | Fluorophore-2 | AGA GAG ACT ACC TCG GAG GCT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44453 | 0.40 uM |
44454 | 0.40 uM |
44456 | 0.40 uM |
44457 | 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 |