Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:54468178-54468260 83bp GGGACTAATGGGGTGGTAGA ACCTCAACCAACCTGTCCTG
Mut = 81 bp
Wt = 83 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGCCCTGGTGACGAGTGGCTAGGGACTAATGGGGTGGTAGAGCCcctagggtggagaggaaggctttatttgaaaatgatagcgcaggacaggttggttgaggtggtggtggaaaaagaaagaagggaggccaggaagggccaatgtcaaagctcagagaggttcatgggggggggggggagggggtgttgtagttttggaaaaagaattagatttctgttttgaattatccagatctttgctggtttcaaggcaccactgtttggataaaagtggcatctggattctgttcttttaaggtcaaagaaaacaactaccggggccacggagatagtgtggaccagctttgttggcacccaagcaaccctgacctctttgtcaccgcatctggagataaaaccattcgcatctgggatgtgaggactacaaaatgcatcgccactgtgaatacgaaaggtgagttcagagagagggggttctggaggaaaagctagtgtgatctgcagcaagcatctttgtagcaagtgcaggttatcaggatgggttgtatgtgtctgtctggagaagacaggaacagaacagagcacttttctggcaggctgtagatgtataggagagcagtggaaactatacaagagtagtatgtgcagaggcgtggagagtttgggagattcaggcaggttacaatggctgagaaaccatggGCGATCTGGAAAGTTGTGCGTTTTGTTTAAAGGCTGTGTTCAAAGAAGCAGTGTGGTAGTTTGAGTAGGCTTGGCCCAGGGCATGGCA
This mutation is a 660 bp deletion beginning at Chromosome 13 position 54,467,578 bp and ending after 54,468,237 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51844 | GGG ACT AAT GGG GTG GTA GA | Common | A | |||
51845 | ACC TCA ACC AAC CTG TCC TG | Wild type Reverse | A | |||
51846 | TAC CAC ACT GCT TCT TTG AAC A | Mutant Reverse | A | |||
51847 | Fluorophore-1 | AGG GTG GAG AGG AAG GCT TTA | Quencher-1 | WT Probe | ||
51848 | Fluorophore-2 | CCG CGA TCT GGA AAG TTG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51844 | 0.40 uM |
51845 | 0.40 uM |
51846 | 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 |