Part:BBa_K3289025:Design

NCBI provides Primer-BLAST for automatically designing primers based on a query sequence. To start designing primers, go to the BLAST homepage and scroll down to the Primer-BLAST option under Specialized BLAST. Enter your target sequence either by cut-and-paste or, if it’s listed in NCBI’s databases, as an accession number. The target sequence is normally found earlier on by searching the particular gene sequence and downloading the fasta file or copying directly the sequence. The ‘Descriptions’ section of the report now includes a download menu that provides direct access to FASTA data, either of the complete subject sequence or only the aligned portion, as well as to other formats including GenBank flat files, hit tables and XML. Checkboxes to the left of each subject sequence allow users to download an arbitrary subset of the matching sequences. Clicking the title of a subject now links to the alignment display, which also contains similar download functions for that alignment along with controls to navigate between alignments and back to the ‘Descriptions’ table. In the "search for primers dialogue box", gene sequences from the fasta file was pasted and blasted against all genomes. The length of the primers sequence was unspecified neither was the genome of the organism specified. The primers were designed according to the preset and optimum conditions for Tm. The selection of the right primers for our experiments were done accordingly to the following parameters : GC content = 50% , self complementarity < 3 and complementarity < 8. Preferred primers had the G-C clamp. The complementarity test were performed using tools from Oligocalc tools.