Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:8935165+8935274 110bp TCTCAGCTTTTGTTTCTCCATC GGTTTGACCCTCAACACTGC
Mut= 108 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TCTTTCTTTTATTCTTTCTTTCTTTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCTTTCCTTCCCCCCCCACCTCTCTTTCAACTAAAAGTCCCCATCTCTCTAGAGCCCTAACAGTGACCCCACattagcaggctgactgggaggaacctcctgtcagtatctttttttgttttgttttgttttttgtttttggttcgagacagggtttttctgtgtagccctggctgtcctgtaactcacttggtagactcagaaatccgcctgcttctgcctcccgagtgttgggattaaaggcatgcgccaccatgcccggctcctgtcagtatcttgtccccacttctctcaggtcttgacacttgatcctggtcttatacttttacctcttctcaatagccgggggaacaaagaccctctgtccagtcctgggggtcccggatctcggaggagcaattataacttaggtatgtaacagagaagtggagggaagaactcgcatgatggaattagtcaggtacggtgggtttgctgctctggccttcaggagggaggcttatttccctgctttttcctgtagaaggcatttcagtgaagatgttccttcgtggccgtcccatcaccatgtacattccgtctggcatccgaagtcttgaggagctgcccagcggcccgcccccagagaccctgagcctcgactgggtgtatcctattcagttccgtgggaatcacccctgcccacaatgggtccctttccttctttacagtttctttctgtgtgggggtggacaaggaatcctgtgaattatcccataccaccctaatgtggatggtgatgccaagtgggagctttgccgttttcatgttcccttcttcctcttcctgtccttcatctggccctttccttgaccatgcccctcaacacagttatgggtacaggggtcgtgattctcgctctaatctatttgtgctgcgctctggggaggtggtctactttattgcctgtgtagtggtgctgtaccggcctgggggaggcccagggggtcctggaggtggcggccagagacattaccggggtcacacagactgcgttcgatggtgagggtcttggggtctggggctggtgggcttgacaggaagagggtctgtctacggggggaagggtagcaaagacagctgccttgctgcttcttgggtggtcttgagagagacccctttcctatagttctcttccccttccttagccttgctgttcaccctgatggtgttcgtgtagcctcaggacagacagctggagtggacaaggatggaaaggtaatgtcaagcagaatatcgagcagataggatgagataacaggtcagcttttcttcccggttctgaccccttctcttccactagcccctgcagcccgtggttcatatctgggactcagagacactcctaaaactgcaggagattggactgggtgcctttgagcgaggtgtgggggccttggccttttcagcagcggtgagctgagcacaaagcttccgatcttgtttgtttgtttgttgttgttttgttttttgggacagggttcctctatgtgtagccttagctgtcctggggctcagtttgtagaccaggttggctttgttttgttttgttttttcgagacagggtttctctgtgcagccctggctgtcctggaactctctgtggaccaggctggcctggaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaagatgtgcgccaccacatccgactgtttgtttttgaggcagggtctcatgtaccttcggctggcctctgtaactcaatatgtagatgaggatgtttttgaactcttgctccttctgtctctgcccaccaattgttataggcacgtgccaccatatctggctagacccttattcttgctcaggacaccactctttgattttaccaggaagcctggggaaggtctagtgttctgggctcacacagccagttctcctgagaaagagaccctcccaacctgtgatagatccctgtctcatagcaggcatgtacagacgtcccggcttcacatgctgtgtctcgtcctcaggatcaaggtgcttttctgtgtgtggtggatgattccaatgagcacatgctgtcagtgtgggactgtagccggggagtgaagctggctgagattaaggtgaggaacctaggtggcttgatggtttctggatctggggtagggtgggagtggtggaatacagacagttccaggtttacttatacatacacttccgactcagacaaactctcagttctctcagcttttgtttctccatctgtgcattgggaactacaggagttctaacGGGGACTATAATTATTTAGGGGATCAGCTCCCCCCCCCTGCAGTGTTGAGGGTCAAACCAGGGCCTTTAATATGTCAGTAGGCATTCTACCATTTGCTCTATACCTTCAGCTTTCAACCAGAGCCTTGAAGGAAGAATTATCTTGAGGTCTCCGGGCTTTTGTAGACCAGCTGTTAGGAAGATTGAGTTATAGAGTAGACTTTGGTGGCATAAGGCTATCATGCCA
This mutation is a 2241 bp deletion beginning at Chromosome 19 position 8,932,972 bp and ending after 8,935,212 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64395 | TCT CAG CTT TTG TTT CTC CAT C | Wild type Forward | A | |||
64396 | GGT TTG ACC CTC AAC ACT GC | Common | A | |||
64397 | TCA ACT AAA AGT CCC CAT CTC TC | Mutant Forward | A | |||
64398 | Fluorophore-1 | CAT TGG GAA CTA CAG GAG TTC TAA C | Quencher-1 | WT Probe | ||
64399 | Fluorophore-2 | AGC CCT AAC AGT GAC CCC AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64395 | 0.40 uM |
64396 | 0.40 uM |
64397 | 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 |