Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:75981236-75981348 113bp TTGAAGAACCTGGAGCAAATG TGACTGGTTGACCCGATG
Mut = 120 bp
Wt = 113 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGGCACTTGGAAGTTAGTTCAGTGTCTTGAAGCTGAAGTTCTGCCTGACCTGCGACACTGTAACTGAAGAATCATGCAAGGATATAAGAAACCTTAGGGAATGAATGATTGAGTCAAGCCGTTGccagagcaacagaatgggatgaggtcgaggcactgcaaccaagttctgtctccatagaggttgttcaagctggacatcaacattcctagagctacgcaccaggatgcgaaagctgtggtctggagggaacgcttatatacagattggccacggtgctctctgatccttaatcctgcctgctctgaacctcataatcagaagacacacccttgaacctagggtgcagttctccagaattttcgggttaagaagggagagtaactgagctgggggagcgggtggagcacagcgctctcatgagtccagttggcttgggcctcaagccatggcgcccaagaagaagcgcggacaaaacgcggggcgcaagcaggaaactacagagcaaccgctctcggagcgcgcgcagtacctacagcgcgagtacgcgttgctctcagaaagcctggtggcctgtgagcagcggatcgacgaggtgctgcaggacaacgagttcctgaacagagaggcgcagcgcctgcgcgaagagaaccggctttatgtcggttacgtgagcgcgcacgctctgcgctgcgccaacgccgtcgtgcgcgtcgaagaccagaaccgcatggatctggcgcagatccggtggcagcgagcggagctagcgtccttctaccacggccgtgaggacggggtgcgcgctcagctgcaggaaatgaagaagcgcgcggaaaacatgacgcagcgggtgcaggagctccagccctacaaggccagtgcgcgcgccccccgaggaccgggcgggctccagagggatgggcgcgctccctaccggtgcgctgatcggcgccccctgctccccaaccctcaccccaccccatgtaggagctgcagctggagcagctggcgcgaatccggaccctggagcgcgagctgctgcacatgcgcgtggagcacacgcagctcctccacggggtgaagcgacgcttcctagatgagaagacggctttcgagcgggaggcgcggctgcaagtgcagtccctgacgcgccgctccgagcgggaggccgcacgttccctcatttcccacgcacaggccctcaaggcggacaacgggtatctccgacaagaactcatgagacttctccagaggacccagttactgcaggacatgcgacagcagctgctagagcagcgggagcagctgcgccgggagcatgtggacttgaagaacctggagcaaatgcacggctggttgcaaaggggccccggggggccaccgctctggcagccaccccaaagcctccaacccagcttgcgcatcgggtcaaccagtcacgttttaaaagcccaggctacgccccagtcagttccaacctcccggggccaatccatggatctgtcacaccgaccctccaagactccatctatattgtccagtgagtctcttccgggcctatctcagaaggctgggtcagtgattgctctccggagtccatcacatccaggatctcgagtctcatctctgactccatcacgcaaggactcgcgtgtctcatccgcgaccccgtcacgcaagggctccagggttccatccatgacctctggtaccttgcggtcgcgcgagggctctcggatctctccacagccctccttgcgtgaggtctctccagaaattgacacacctgcaaagtccagttccaagcttcccacagccctgttggaagatcaggcccctctcagcccacagttggaggaggcagaaaacgctgaagacgacacagaaactgtcttggagcaagcctgaacccacacgaggaggcaggccagtagggcatagggtggaggacaaaccttggaagatttcttggtgcatagatatgttaataaaagtgttaaataagctctgtgacgctctgacacttgcctgggtcgatgcccttgtatttcaagtgagcagaggcctgctattcgagctacagcagagctggttgtatccttggctgctggaattcgatgttcttccaccaacagccagcaaggcaggcaggcttagggaaagttgggtagtgtaatttcagcagcagagggcagattctgttctcagcacagggagaagttactgggcttttccaatgctttatataatacgcacacacacatatgtatgtaggtatgtatatgtatatatgtatatacattccaatttagggaacactgcaagaattattaggaattcccattgccacccatctagatgaacagggatgcatgttatatatttggtactttcagccatccgggattctgttggagacagttatgccccttaattttaaatatatccctgtgtttactgaataggactttgacctgttttcttccggtgctgggtgttgaacccagggctacctcctgagctacagcccaagccctgtcccaactttcttcaagacatgattaaaattaggaaatgcaccttggagaaaatactgtaaactaaatcatactggccaaattgtccctgtggtgtctgtagctagctatggatcttacctgggtcacatcttacattccattgtgctggtctgtcctttcctttatagaaagggtctgtataggtcaggctggccttgaatccaagagatcctcctgcctctgtctcttcagtgctgagattaaaggcatccaccaccacctcagccccgttgtcttgtctttttttttttaaacttctttttaaagatttatgtatatgagtgcactgtaactgtcttcagacacaccagaagagggtgttgggtcccattacagatggttgcgagccaccacgtggtcaccagggattgaactcaggaccactggaagagcagtcagtgctcttaaccgctgatccatctctccagccctctttgtcttaatgtacaagaccagtcGTTAGAGCACTCCTTAACTGAGTTTTGGAGTCTGTTGGCTTTGAGCACCTTTCCTGTCCAGGAAGTCCAGGGCAGTTGACACCTCCTGACCCAGG
This mutation is a 2917 bp deletion beginning at Chromosome 15 position 75,979,638 bp and ending after 75,982,554 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52088 | TTG AAG AAC CTG GAG CAA ATG | Wild type Forward | A | |||
52089 | TGA CTG GTT GAC CCG ATG | Wild type Reverse | A | |||
52090 | TGA CCT GCG ACA CTG TAA CTG | Mutant Forward | A | |||
52091 | AAG CCA ACA GAC TCC AAA ACT C | Mutant Reverse | A | |||
52092 | Fluorophore-1 | CCA AAG CCT CCA ACC CA | Quencher-1 | WT Probe | ||
52093 | Fluorophore-2 | AGT CAA GCC GTT GGT TAG AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52088 | 0.40 uM |
52089 | 0.40 uM |
52090 | 0.40 uM |
52091 | 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 |