Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 98 bp
Wild Type = 116 bp
>chr3:89248340+89248455 116bp TCTTTGCCTTCCTTTCTGG TCCCATGACTTAGGAAAGAGG
CACAGGTGACTCTAGGCCATGGGATCATGACTGAAAGATGAGTGCAGCTTGTCCAGAGAGACAGCAGATGCCTTGGCTACAGAAGGCTGGAGACCAAGCAGGGCGGTGGCCTAGAATGCTCTTAGAAGCATTATTTCCTCCTTGCCACAAGAGGGCGCTGCACCCAAGGCGCGTAGAGGAAGAAGTAAAGCAAGTCCACGTTGGCCCCGACGCCTGCGT[acagacaaccgcgccggctttggctcccctctttctccagggacacggattggaccacggacggggcgtgatgggtaaccgcaccggcgggatgcccgggctgctctagcgggagtgggcgttggggtggtcctcttttctccgtagcctcggtaaagctgacgtgtcctgggaactagcatttcctccggacaggcgggaacttgggtgtgcagggccgggtggttcctggaggtcgctgtggggtgggggctgggtcacttggaagtggttggtgagccaagctccggagtgaatctgcctgaatcttagcctgactctttttctcatttatagtagtcggtaccgggacctccttggcgctgtcctccctcctgtccctgctgctcttcgctggcatgcagatctacagccgccagctggcctccaccgagtggctcaccattcaggggggcctgctgggctccggccttttcgtcttctccctgactgtatcttcctcgcgctggaaggggaggcgggggtgtatgaacagaacatgggcgggagtgggtggcaagggctttgcagtccctctacttcctatcttgttgcgtgagtttctccttaattacatcacccaggccttcaataatctggagaatctggtctttggtaaaggcttccaagcaaaaatcttccctgagagtaagtgaaactcaatagttaactggactcttttttttttaaacctctcccaaacatgatgccgaatttccctgtgcttagatgataaaggagaaatacatttgcttaacacaatcttgaagacagaagatgctttcagattaagcattttattggagctggaagagaagcacaaaaaggggggaggggaggggaggggagggagaggaaaagaaagaaaaaggccaacctggtctgctctcaggacaggtttcaagtgaacagtcaagcctgggctgatgtctgataggcatccaggagaaggtgttctttgtccctgtgagagaagaaagcaaaaccaagcccactgagggattctgatgtggaactgagcccgcactattgactgctgctggaggagattgtgaagtctgtgttcactgggatgttaccaatagcaggacccctgacagaaggagaatagagaataaccgctagagctcactctctttctctgcatccgcttgtttctctcttcctttcttctcctcccttctgtagcccacactggccttgaacttcctgtgtggctgtgaatgactaaacccttgacttgctgatcttcctgcctccacttcccaggtcctgagattagcactacctcatgtagtttatgaggtgctggcgattaagcccaggtccttgaacatgctaggcaatctctctcccaatggaggcacatctccagccctgatagatcagtttttgtgtgtgataattcttgttagatggttcatatagagatctgatggagaatagggctcataaaacaaagtcggcatcaggaccgagccacttggaaagccacatgctgggaaaagcaagcagttgtggccccgtgaaagactcacccagaaaccccgaagcccgccattgttgctgtcttcacggccctgttggaggtttccagagcttccgcccagcaatgcccagagcttctgtctcttccactctgtcccatgtgtttcttatatgctgaaagaaattgttggaaggcaaaaaccttgtctttcagccagacagtgatggcacaggctcttaatcctagcactagaggcagagggaggcaggtctctgtgagttcagggctagcgcgatctacagagcaagttccaggacagccagagctacacagtgaaacctgtcttgaagtgagaagaaaaaaatcctcttctttgccttcctttctggctccagctggacaagtggacctcttagccccttccaccctctcatccgtatcctgtcctcttagagaagccataatcctctttcctaagtcatgggaacacgcctgctttccgcccttctagaacattttatttattgatttacttttgaggcgggatctcactatgttgcccaggctggccttcccactgtgtagcccaggctgtccctcccttggattcctgggcttacgtgatgcttcctgccttatcctccggactggcttgctgtgtaggcatataccactgcgttttcgctttcttctgtgtatgttcgggggttgggggaccaggaagttaaatgaaacgtggagccaagctagtcaaacaaaacgccattcatatcttcccttggtgtcctgaacaagtgttcaggcacaaggcatttgaaatccctgcttttattaaggcatacctattttgtttgtgttgtttttctagtcaggatttctctgtgtagccctggctgtcctggaactccctctgtagaggaagctgacctccagctcacagagatctctctgactctgcctctagtgctggggttaaaggtgtatatccccatgccaggcaggacgtacgttttcaagcaggttcctcacagggttctagactgctcctacttttccgttgctcttttgaccgccttctgattgaccctcttcctgtctccccagttctcctctgcttgctgttggcgctctttgcctccggcctcatccaccgagtttgtgtcaccacttggtacgtatctgacagctgggcaagcggtgcaacaacaccatgtcttgtgggaccagatcttcctatggtgttaatagtcccttcccttcccacc^GGCATATGGTGGGAAGGGAAGGGACTATTA^]ATATGCCACCTCCTCTGTCCCCTCCCCCCCATTATTTTTTTCAAAGCTTTTTTTATTTATTATATGTAACTACACTGTAGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATCTCATTACGGATGGTTGTGAGCCA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46251 | AGG AAG AAG TAA AGC AAG TCC AC | Mutant Forward | A | |||
47717 | GGA GGG GAC AGA GGA GGT | Mutant Reverse | A | |||
47718 | TCT TTG CCT TCC TTT CTG G | Wild type Forward | A | |||
47719 | TCC CAT GAC TTA GGA AAG AGG | Wild type Reverse | A | |||
47720 | Fluorophore-1 | CCC TTC CAC CCT CTC ATC CGT | Quencher-1 | WT Probe | ||
47721 | Fluorophore-2 | CGC CTG CGT GGC ATA TGG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46251 | 0.40 uM |
47717 | 0.40 uM |
47718 | 0.40 uM |
47719 | 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 |