Part:BBa_K3289017:Design
pcAa RPA Fwd
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Genes were located in .fasta format on NCBI and sequences were created on Benchling. For RPA primers generated from Primer BLAST, the following values were used in settings according to TwistDx recommendations. Product Size: 100-200 Primer Melting Temperature: 50-100 Primer Size Range: 30-36 Primer Size Optimal: 32 Primer GC Content: 30%-70% In addition, a Python package called primedRPA (detailed in the paper titled PrimedRPA: primer design for recombinase polymerase amplification assays, available at https://www.ncbi.nlm.nih.gov/pubmed/30101342) was used. The following parameters were used: Desired primer length: 32 Desired probe length: 10 Desired amplicon length: 100 Minimum GC content for primers and probes: 30 Maximum GC content for primers and probes: 70 Tolerated length of the region which could form secondary structure: 5 Tolerated number of background binding nucleotides in primer or probe: 24
Multiple primers were generated and then annotated in Benchling and optimal primers (preferably 1 pair from primer BLAST and 1 from primedRPA) were selected based on location, GC content, fitness within PCR amplicon of the same gene, and quality of available gRNAs within the amplified region of the gene itself as well.
Source
This part was synthesized directly from IDT.