Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:21593727+21593821 95bp CCAGTACAGATGCCAATAGGG TCCTTCATCCTCCACATCAAC
Mut = 105 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTCTCAGATAACCAACAACTAGTATGGACATGACTGGACTTGAAGCTGAGACTTCTCTGGGCTTTGTAATGTCTGCAGTCGGCTTTCCCTAAAGCCTCATGCAAACATGGCTGATACTCCGGGGTTTACAACAcactgcggcaggggaggggcgggacttccagggcggaattttcggtgacatcaccacagaaagttcgttaaagcaagggctttggctgcgttctggaagtagggcccgaggtctgtcggtggggtggcttctgcagcgtgtggccgtgtgaccccacggaggtgacctcccaggtgagtcctccgcgtccctcggtggtgatcagtgtgtcgtcctgagatgtagtgtcgtggatacccagtgtgccctcttctctcgtctcttccacagagcggctgaggttgagaacccattgagcagctaggccccagtgtatgcgctgatcctatggcggaggcccgtgggaaccaaggcgagagctgtgtgagcctggaggacgtgtgcctgcgtttcactgcagaggagtggggtctcctcgatgacactcagaagctcctgtaccacaacgtgatgctggagaatttcttgcttgtgctgtccctgggacttccgatttccaaatccttcccaggtaccccagcgatagatgctgtcttagtcagggtttctattcctgcacaaacatcatgaccaagaagcagttggggaggaaagggtttattcagcttacccttcaatactttctctgtgataactccagctgtgtcaagttgacacaaacccagccagtacagatgccaatagggactccctgttaaccaaagcatgatggcgtaggtaatctaagctcgcTCCCGAGGTTGATGTGGAGGATGAAGGATGTGAGCAGAGCATCTTCAACAAATGCTTCACACCCTGAAGCTTAGCTCTGTAAGAGTGTTAGGCATCAAATAAAAAATTTAACCAGTTATCCAGCCTGGAGAACCCAC
This mutation is a 732 bp deletion beginning at Chromosome 10 position 21,593,062 bp and ending after 21,593,793 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50572 | TCC TTC ATC CTC CAC ATC AAC | Common | A | |||
50573 | CCA GTA CAG ATG CCA ATA GGG | Wild type Forward | A | |||
50574 | CTG GGC TTT GTA ATG TCT GC | Mutant Forward | A | |||
50575 | Fluorophore-1 | TCC CTG TTA ACC AAA GCA TGA TG | Quencher-1 | WT Probe | ||
52220 | Fluorophore-2 | CTT TCC CTA AAG CCT CAT GCA AAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50572 | 0.40 uM |
50573 | 0.40 uM |
50574 | 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 |