I have designed a set of PCR primers for my fusion genes of interest. Now, I want to design a probe for executing ddPCR. Kindly advice about what are the necessary tools I can access online for this purpose.
Bioinformatics Tools:
BLAST: To identify the target sequence and check for homology with other genes.
Primer-BLAST: To design specific primers targeting the fusion gene sequence.
OligoAnalyzer: For analyzing primer and probe sequences to check for secondary structures, dimers, and hairpins.
Molecular Biology Tools:
PCR Primer Design Software: Tools like Primer3, NCBI Primer-BLAST, or IDT OligoAnalyzer for designing specific primers.
Probe Design Software: Software like IDT OligoAnalyzer, Integrated DNA Technologies (IDT) PrimerQuest, or Thermo Fisher's Primer Premier for designing probes.
PCR Master Mix: To prepare the PCR reaction mixture, including enzymes and buffer required for ddPCR.
Fluorescent Probes: Probes labeled with different fluorescent dyes (e.g., FAM, HEX, VIC) for detecting the target fusion gene.
Digital Droplet PCR System: Equipment required to perform ddPCR, such as the droplet generator, thermal cycler, and droplet reader.
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