Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 77 bp
Wild Type = 72 bp
>chr15:79643657-79643728 72bp TGAAATAATGCAGTTGAGTAGGAA AGGCTAATGCGGAAGAGGAC |
Wt Sequence (deletions in lower case):
ACACAATGGGACCATGTCAGTGATGGGTGGCAGAGGCCCTTACCACCagcgggtagaagagaggtgcttagctacacatgttgctccaggcttgcaggtgccaataaggaaaatgctctattccctgaccctcgccctcttccctggttacatcaatttcttatgcccacttttcctcctcctcctggtttttgaaggcaattgaagactatgagttggagaagaagtctttaagagagaaaactcacggcagcccaggagacaaaggcaaaaatattttctcagcctaaaaacgtgtttttgtggggtttgattttatttttaattttctttagtttgcatacttcttagctgtttctgttttgtggtgctaggcatggatcccatggccttgcacacaccaaccacatgccctgtcactgaggtggaccccggtggggtttttctgtttgtttgtttgtttgtttgcttggttttttcagtttttgaaataatgcagttgagtaggaatccagagctctaccccagGATAGGTTTTGTCCTCTTCCGCATTAGCCT
This mutation is a 481 bp deletion beginning at Chromosome 15 position 79,643,687 bp and ending after 79,644,167 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66014 | AGG CTA ATG CGG AAG AGG AC | Common | A | |||
66015 | TGA AAT AAT GCA GTT GAG TAG GAA | Wild type Forward | A | |||
66016 | ACA CAA TGG GAC CAT GTC AG | Mutant Forward | A | |||
66017 | Fluorophore-1 | CCA GAG CTC TAC CCC AGG A | Quencher-1 | WT Probe | ||
66018 | Fluorophore-2 | AGG CCC TTA CCA CCG ATA GGT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66014 | 0.40 uM |
66015 | 0.40 uM |
66016 | 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 |