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It's gotta be either nonspecific binding or the polymerase (we use TaqMan). According to the ncbi primer blast my primers are a 100% match to the exon and have an off-target recognition with 5 bp or more mismatch, so I'm not sure why it's the case.
Here at NEB, we have created a variety of interactive tools to help you accurately design primers to suit your specific needs. Select the application to get started.
I'd recommend NcBIs primer BLAST. Put in your target gene of intrests sequence (can find FASTA sequence right on NCBIs database as well) and make sure you select the organism you are planning to amplify in. Will give you a readout of a few primer sets.
8 lis 2021 · PCR primers are interdependent with your sample type, target, reagents, and thermodynamic conditions. Establishing your PCR objective can guide your primer design and enzyme choices. It’s a good idea to review parameters and understand why primer design is such a critical, dependent variable in PCR. Primer melting point
Try to find a stretch of 20 or so bp for your primers with quite equal balance of ATs and GCs. If you're designing probes, make sure they anneal first to your amplicon by making Tm difference of around 3-5°C from your primers. Blast your primers to make sure they aren't non-specific.
We choose 15 positive and 5 negative nasopharyngeal and oropharyngeal swabs from patients with acute respiratory tract infection. The samples are tested with DeCoV19 Kit Triplex, GeneFinder COVID-19 PLUS RealAmp Kit, and the AI-designed primers UtrechtU-ORF3a.
Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets.
