Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:77691431+77691566 136bp ACAACAAACCCTACAATACAGCT GCATTACCGTTTATTTCACCATAG
Mut= 136 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCTACTGGCCCAGAAGATGGACCTGATATGAGTTGTGTCCTAGGTCTGCCTCTGCCTAGACATTGAACCCTGTATCATAATCTGTAAAAGTTTCTTTACATAAAGTTTCTTTACATAAAGATAAGATACAACAAACCCTACAATACAGCTTAgtattgggttcagtaaagttgtttggtcttagtactactgtgatttaaatgtatgtgttttgtttgtttttgttttccttctataggctatggtgaaataaacggtaatgctggagaaagagaaatatctttaaagagcctcagttctgatgaagctaccaaccctatttccagggtccttaatggcaaccagcaagttgtagaaacaagcctgaagcagactgtaaagaccagcacctttggaaaagcaggaatcaaaaccaagaattttattcagaaaaacagtatggacaaaaagaatgggaagtcttatgaaaacaaatctggagagacccaggctgtagataagaccgataccatagcaattccaaatggtgttataacaagtagttcaggctatattactaatggttatatgagcaaaggagcagataatgatggtagtggatctgagagtggatataccactcctaaaaaaaggaaagctaggcgcaatagtgccaagggttgtgaaaaccttaatttagtgcaggacaaaataatgcaagagactagtgtcccagcattaaaacagggacttgaaactttaaagcctgactatagtgaacaaaagggaatgcgagtagatggttcaaagcctatttggaaatatgaaactggacctggaggaacaagtcgtggaaaacctgctatgggtgacgtgcttcgtaaaagctctgatattaaacctggtttgagtagcaaaaaatttgatgatcggcctaaaggaaagcatgcctcagctgctgcctccaaagaggactcatggaccttgtttaagccacccccagtttttccagtggacaatagcagtgcaaaaatagttcctaaaataagttatgcaagcaaagttaaggaaaacctcaacaaaactgtacagaactcctccgtgtcaccatcttcatcttcatcatcatcatctactggggaaactcagacccagtcttcaagtcggttatcccaggtcccgatgtcagctctgaaatctgttacttctgccagtttttctaatgggcctgttttagcaggaactgatggaagtgtgtacccatctgggggtcagccactgctaacgactgctgctaatactttaacacccatctctactgggactgattctgttcttcaggacatgagtctggcttcagcagccgttgaacaaattaaatctagcctttttatctacccttcaaatatgcaaacagtgctgttaagtgcacaagtagatctgccctctcagacagatcagcaaaacctgggggatatcttccagaatcaatggggtttatcatttataaatgagcccagtgctggcccagagactgttattggaaagtcatcagatcataaagtgatggaagtgacatttcaaggagaatatcctgccactttggtttcacagggtgctgaaataattccctcaggaactgagcatcctgtgtttcccaaggcttatgagctggaaaaacggactagtcctcaagttctgggtcacattctaaaacctgggactactgagagtggagccttatccttggaccccagtcatataggtgacctgcaaaaagcagacaccagtagtcaaggtgctttagtgtttctctcaaaggactacgaaatagaaaatcaaaatcccctggcctctcctacgaacactttgttaggctccgccaaagaacagagataccagagaggcctagaaaggaatgatagctggggttcttttgacctgagggctgctattgtatatcacactaaaggtaactcatttgttttttttctaagattccaatgggctgattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtacatgtatatttgatattctatattatgaatgactgggtaagaattagtatcaattaattgttaattgatttgaaatttttgaaaatttattaaagtttagaggactgtagttttccaacgtaaaatcatcaataaaggcacagttgatcacataattagatagaggtttcatgtcccttaaaggatagcctttttttgtaTTGGGTCTCAGGTTTTTTTTTTTTTTTTTCTTTCTTTTTTTTAAGTGATAGAGTCAAAGTTTTAAATTACCCAAATGGGCTTTAAATCCTGATCCTCTACATACTAGTTGCTGCTGGGATTGCCTGTGTATGCCACCAAA
This mutation is a 2080 bp deletion beginning at Chromosome 11 position 77,691,456 bp and ending after 77,693,535 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44010 | ACA ACA AAC CCT ACA ATA CAG CT | Common | A | |||
44011 | GCA TTA CCG TTT ATT TCA CCA TAG | Wild type Reverse | A | |||
44012 | GCA ACT AGT ATG TAG AGG ATC AGG A | Mutant Reverse | A | |||
44013 | Fluorophore-1 | ATT GGG TTC AGT AAA GTT GTT TGG | Quencher-1 | WT Probe | ||
44014 | Fluorophore-2 | AAG TTT TAA ATT ACC CAA ATG GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44010 | 0.40 uM |
44011 | 0.40 uM |
44012 | 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 |