Primers of all genes in Bacillus subtilis strain 168 without recognition sequence of NdeI and BamHI

Primer design is a critical part of the molecular cloning workflow for amplifying target genes from a template. Serving as a molecular marker for the region in which polymerase chain reaction should amplify a particular gene segment, primers should have perfect complementary base pairing with the target strand of the DNA duplex in which the gene resides. Beyond the binding sequence that anneals a primer to the target DNA strand at the correct location, recognition and flanking sequences are also typically appended to primer sequence to aid downstream restriction enzyme digestion for molecular cloning purposes. Specifically, recognition sequence appended to the primer facilitates recognition by specific restriction enzyme for digestion and excision of the gene fragment that is subsequently inserted into an expression vector. However, one critical requirement for the choice of restriction enzyme is that the gene of interest should not contain recognition sequence of the restriction enzyme. In this contribution, a primer database for all genes in <i>Bacillus subtilis</i> strain 168 (Genbank ID number: NC_000964.3) that do not contain recognition sequence of restriction enzymes, NdeI and BamHI was obtained using an in-house MATLAB software processing the annotated genome sequence of <i>B. subtilis</i> strain 168 from Genbank. The database comprises the gene identities together with the forward and reverse primers necessary for amplifying and inserting the gene of interest into an expression vector. NdeI recognition sequence was appended to the forward primer, while that of BamHI was appended to the reverse primer. Melting temperature of the primer was designed to be 55 ^oC. Two nucleotide flanking sequence of ‘AT’ was appended to the start of the forward and reverse primers to help anchor the RNA polymerase during transcription. Altogether, the database comprises 3202 genes that are without recognition sequence of NdeI and BamHI. Collectively, a primer database comprising genes in <i>B. subtilis</i> strain 168 that are without recognition sequence of NdeI and BamHI restriction enzymes was created using a MATLAB algorithm. Both forward and reverse primers sequence are available in the database and should find use in workflows attempting to clone specific genes from the <i>B. subtilis</i> strain 168 genome using NdeI and BamHI for the forward and reverse primers respectively.<br><br>