Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:53452568-53452713 146bp TTTTAGGTACAGCCCAAGT AGCTTCTCAGTCAAACAGGTTGC
Mut= 162 bp
Wt= 171 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTGTAGACCGGCTGGCCTCGAACTCAGAAATCGGCCTGACTCTGCCTCACAAACGCTGGGATTAAAGGCGTGCGCCACCACGCCCGACTTTACCGGGATGCATTTCTGCTACTTGTTTGTTTACTATCTTTTCAATTGTCCTCTTGTTTTATTTTAGTAGCATTTGTAGCGTTTTAGGTACAGCCCAAGTTTAAAATACATTCCACTCCttaaccaggctttcttgaccctgaaatgatcaacgctttttttccctctaaggcaggaaaagttaagccagttggatacagagttgcaacctgtttgactgagaagcttcccagggtgagcacgtcccagtcttcctgtttgttcctcaaatatgagttgtagggagactagattctcagctttctttatgaaagggtgcacagatgtaacgtgtctagttttgaatctttactatattttggcgttatctattgatccatattcatccaagaaaatgaaaatgaaaataaacatttttagtttaccaaaaattctaagagtcaatgtatttcagtaggaattatgggatacttgtaacctggatgtttggggggggggggagcgggaggagggtaggggatgtctgtgtgactgtaagtcacatgtgcgcaggtctccatgggagccagaaaacggcatggcatctttggcactagagtgctagatggttctgtgcaccatgtgggtacagggaacccaactcagggcccctggaaaagcaacaactgctcgtgtaactacttagtcttttccagtcccctacattatagtcttaaaaatgttttaaaattaatttttattttatgtgtatgagtgctttgcctgtacttatgactgttcaccaggtacatacctggtgcccacagaggtcagaatggatcatcagatcccctggatctggagtttcagagcaatgagccaacatgtgggtactgggactgaacctgggtcctcttgcaggagcaacaaacgctcttatgcacttagccatctcttcaggcccccaaactataatatttaaagtatttttttccaccagtagacacagtttagatggtgtgtcctagttttcagatgcaggaagtcatcactggctagggaagctgagttgatattttgaaactcatgattcatatgtctctttgtctcactttgcccagctaatgactcccccagaggcaaagaagtatttcaacttcagatatccacctgctggagcagaaagagtattttatggcagagcaaatgacccccaaattgcaccttatttgacacatggactaagatctaaaatctcaataccggtaaacctttctcttttttttcctcctacattaatcattcatttacgaagttagaaagttgtacaaatcaaggtgtaagtgaaattcataattactatgggtattttccttcctcattctggcttctaaaaacactagtcttgggcaatgggttgtgttttcaagtaactaaggataggcgaaggaagcagcagttcaaacccagagtttactgatcctcagatgggatgaggctgcgacattcccattttcccagtaaagaaaaatgttggaatatcttattagccattaagcgaataggcctgctccttttgcccaaggccctacagagagcatatatagatcttgagtggatcagaaaatatttgatgctgactgctgggatataactgtcctctaattgcttatttatctaccttcctacctttccttctatatttgttatctccttttatttatttcagtttattaagtttatttatttagttatttactggtggggctaaagctggaactcggggtaatctgtattctgggtaaacactactgttgagacctatcatcaaccctctatctttttgtttaaaaaccttttcccacattttgtgcgtgtgtgtgtgtgcgcgcgcgtgtgtacatgtgctacagcacctgtgtgggggacagatgagaacttgggacagtcagttctttccttctatcttgtgatttctggagactaaactcagatcatcagccttggcagcaagctcccttacctgctgagccatcttggccattaaaaaaaattcattgattaatttttagcgtgtgtgtgtgtgcacatgcacgcatgtgtgcacgcacgtatgtgttttatgtgtgggcatgcatgtagggacttagaggacaactttgtagagttgattccctctttctttacatggtttctggggatcaaagttacactggcaggctcttgtggcagtatttttacttgctgatccatgcctcagggccttttctgtttttgagacaatgtactatgcccagcatctatctctcttgtctttctcttcctgtctatctctcccctgctttttgtttgtttgtttgtttgtttgaggcagtgatttttctacatagccatagctatcctggaacttgctttgtagacaggactggccttgaactcagatccacctgcctcttcctccacagttctgggattaaaggcatgtgccacaccatgcccagtgctttctctcatttttatcttttgttatttgaagtttttaactctgtagtccaggttggcctcaatcttcctgcctctgttacttaaggactgggattacagtactgtgtcattaggtctaatctgggttttatatgtttgagaaagttggtggaaatgattttatgttctgtgcttgtcatacgtgttcttgagtatttgcatcaataacctggaaactccttccataagtcgttagcaccactgtaggaaagttaaatttatctgtctgtgtatgtatgtatgtatgtatgtatgtacatatgtacatacttaactatctatttacttaatgctggggattgaacccgtggtcttgtacctgttaggcaagtgctctattgctgagctacttctctagtccttttttattttctgctttgtggttagaacttacagcctcatgagcactccactactgaaccattctccatctggtcttgcaatctttctgtagtttcatgcctgcctggagcttagtatcctggtacttcagttcccccaagagctgggatacagtcctagctgaaagttctgcatctgctttgttaaacttttcagtactgatttcattgtcagttcattggaagatgagcacagccaatgatgatgggggaaatagcactttaatcgtattttcttctcattagagttttagttttcttacaaatactggtttcgatacatgaacattctataatttctttagatgggatcattgataaacccacagcccattaccacgtttcaacagaaaattaaagacaaaaaagaatctatatattttagccatcaacgagcaccattgggaaaatctcatgatcaaacaccaggattgccaaaaggaatggatgtgatcaacacaacattagggacaccgactatcagaggtagtaaaaataagggacaggaggaggtgggaaatacgtcttatgaaactggattaaatatgtcactgatatattttctgttgatgatgtgaaggattgttagtgatgaaagatgaacatagtcttcacacaaggtcagtggtctgcttgggccattctctccttctatcttgtcctgtTGACAATGACATCACATCCTCTCTGTCCCCTAACTATAATCTCACGTTTGCATTTTTTGCTTTCAGTTTTCTGCTTTTATTTAAATCTATACTTTTTTTTCTACTTAGTGGGTATCTTGCCATGGTACCTGAAGAGTGTCGCACAGAGAGAGATGACTGAAATACTCCAGGTACAAGTAAAAAGATGGTATCATAGCAGGATCTCTCGCT
This mutation is a 3470 bp deletion beginning at Chromosome 17 position 53,449,206 bp and ending after 53,452,675 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48285 | TGG CAA GAT ACC CAC TAA GTA GA | Mutant Reverse | A | |||
48286 | Fluorophore-1 | ACC CTG AAA TGA TCA ACG CTT | Quencher-1 | WT Probe | ||
49191 | Fluorophore-2 | ACA TCA CAT CCT CTC TGT CCC CTA | Quencher-2 | MUT Probe | ||
49281 | CGT TTT AGG TAC AGC CCA AG | Common | A | |||
49282 | ACT GGG ACG TGC TCA CCC T | Wild type Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48285 | 0.40 uM |
49281 | 0.40 uM |
49282 | 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 |