Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:54262556+54262662 107bp AGAGGAGAGAAAACTGAAGAGTGT TGTTCTGGAGACCTGGGAAG
Mutant= 95 bp
Wild Type = 107 bp
Wt sequence with deletion in lower case
TTCCTGAGATTAAAATATTGACCAAATCAAACTCGGGGAGGAAATGTTTATTTGGCTTACAGGTTACAATCCATCATGAAGTTTAGAAGCCAAGGCAGTAACCTAGAGGCAAGACCTGAAGCAGAGACCATAGAGCAATGTGGCTTCCTGGCTTGCTTCCCATGGCTTCCTCAGCATGTCTTCTTTTATCATTATTATTATTATCATCATCATTATTACCCTTTTCTTCTGAGACAGGGTCTTTCTATGTGGCCCTCGTTGTCCTGGAACTCACTGTGGAGACTAGGAGGGCTTCCTAAAATGATCTCTTTAAGAGGAGAGAAAACTGAAGAGTGTATTCTAGGCAAAATGTCTGTgctctcaggaagcattcttgatgctttacccaggaattcttcccttcccaggtctccagaacaagttcctggcccgttatgtatccctcccaaaccagaacaagatctggacggtgactgtgagcccagaacaaaaggatcgcacccctctggtgatggtacatggctttgggggcggtgtgggcctctggatcctcaacatggattcactgagtgcccgccgcacacttcatacctttgatctgcttggttttgggcgaagctcaaggccaacattcccaagggacccagaaggagctgaagatgagtttgtggcctcaatagagacatggcgggagaccatgggaatccccaccatgatcctcctggggcacagtttgggaggattcctggccacttcttactctatcaagtaccctgaaaggtaacttaagctactcttctcactcatgagctgactctgccttcctacacgttccaatatttatcccagtaatgctctcattccccagcaactggaaaactatgctacctccaagcaaatacagtgtatcttctgtttaatgtagagttaaacatcttatcctggtggatccatggggctttcccctacgaccaactgaccctagtgagatccgtgcacctccaacctgggtcaaggctgtggcatctgtcctgggacgttccaatccactggctgttcttcgagtggctgggccttggggtgagtcacctcccgatgaaggactctgtctttaagccaaggccatccaattcaccaccactcaccttggcgcacctacctatctctgtcatcctcactggttcttaaacactaggcctgcacatagcattgttccttaaaaccattcatcagaaccatgtctctgtattggaggtttcaggtgattgagatatatatatatatatatatatattcccttcatctgtatcagcagatagaaccagagaaaggatatatatatatatatttccttcatctgtatcagcagatagaaccagagaaaggatatatatattcccttcatctgtatcagcagatagaaccagagaaaggaagggactctgatgtgccacttctctgctacatgagtgaattttgtgtgacaacctggtagagactcagggcagtagccaccattggatccctttccatggctcaccagcaccacagcaagccggactcagatcctcagcttctaacataacacaccatggcagacaggtcccactgagccatgccctgctccagagacacaccgcatctccctctctggatttcttagcactaagtagctgtaagaagattctgagatcgagacaggacacactatagtctgtagtgtcagaggctctgcatgctctcacctcacgctgtccacgtgcctccctttgaggagcttttgacactgccgacataagaaacaggtccatttgactgaaaaggtggtgacccctgttttgtacggatagcttatgtctacatagaacttttcagtagtcacttgttagactggctgaaacgtaatggcacctaacagagtggaaaatgagccctgacatataaggtagcacaggtgttctgggataaagggagcaagtaagagacatgggtgccaaagggaagtaggcagccagagggctgacctcagagtccagggccctctaacagactcactaccatccttcctggtctaaccatgctcccctcccctggtcttggatctggccaggtccatgcaaggtatcagctatggtgttgggtagctgcctggcagccatggaagactgtgctagagggacagcagggcctgcggctatattcatggtatctcccccagatatcgctcacagacaaaaggcctgccaggacagaggcagtgtccagtgggcccggtcctcacattctgcctgcatgcctgccgcttcttcacgcacagtggtgaaagccggggccttcagggctgttaaggtcacctttccaagtattctgttacagcccgaaagtgagagagtgcttgggaaatggtatggtttcattgctaccttcagaaaagtaaaaataaataaataaataatcgcaaggttacagggtggggttggggtggaggagggggaatgcataactctgacttgaaaatcctctcaaccacagaccctcccagaacgtgtgagcctgtaaggaaagtgctggggctagctgacatgaaaattaccaacttccgtggggatggtagactgtcactaaactcattggtgaccaaaaaatcaaactctgacatttgaagtcatctgttgcttcccaacaggctcctgcactctctgtcatgttgactgcataaaggaactaagttcgtcaagagatgaactgggagaaggttgtttaaacataagcgggcaggagttcctcttctctccctgccaccaccactcactctgaccgctccacggttttgtttgttttgttttttggtggtggtggtgggggatgttgttgttctttctgttctctggtaggggcttaaaatagggtctcacacatccccagaccgaccttgagcttgaaacagttctcctgcgtctgcctcctgagtgctggaatcccaggcacgtaccacttgcttgcccagtatcacgttgttcttcctaccccctcccagggcctgggctggtgcagagattccgtccagacttcaagcgcaagtttgcagacttctttgaggatgacaccatctcggaatacatctaccactgcaatgcacagaatcccaggtgagggcggtgcccaggaccagtgcggggtggggctggggaggggctgaaaagtaaggctttgagctactgcttctcttccacagaagcctgcctcgggggaccccaggcaccagcaggcatgtctccagagttccctaggggtggtcaccagagctctaggagacaatagacttaattaaatatgctgtccctctctagaggaagacctcatgGCTTCCCTAGCGATCAGTAGCTGACATGATGAGAAACCCTCTGGGAAACATTTAAAAGATAGTTTATAACTGAAAAACTGATAAAAAAATTTTTTTGGTGTTTTGGGTTTTATTTTATAGGCAAATGTGGGGGGGGTGTTTTTAAGGCAAATAGAAAAGTGAAGTCTTTGCCCATTCACAACCTTCAGTTTCTAGATTTTCTTATTTTTAGTCTAATGTTTCATGCCATATTGATCCAGAAAGACTTCTCTCATGTGTCCC
A 3008 bp deletion beginning at Chromosome 14 position 54,262,600 bp and ending after 54,265,607 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44793 | AGA GGA GAG AAA ACT GAA GAG TGT | Common | A | |||
44794 | TGT TCT GGA GAC CTG GGA AG | Wild type Reverse | A | |||
44795 | ATG TTT CCC AGA GGG TTT CTC | Mutant Reverse | A | |||
44796 | Fluorophore-1 | CTC AGG AAG CAT TCT TGA TGC | Quencher-1 | WT Probe | ||
44797 | Fluorophore-2 | TGT CTG TGC TTC CCT AGC G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44793 | 0.40 uM |
44794 | 0.40 uM |
44795 | 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 |