GenElute™ Bacterial Genomic DNA Kit

Protocol tips

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	The Proteinase K solution must be added directly to each sample every time. Do not combine the Proteinase K Solution and Lysis Solution for storage.

Protocol tips
The Proteinase K solution must be added directly to each sample every time. Do not combine the Proteinase K Solution and Lysis Solution for storage.

Publication protocol

Genomic DNA of the different bacterial strains was isolated using the GenElute Bacterial Genomic DNA Kit (Sigma) according to the manufacturer protocol. The genomic DNA was subjected to several PCR screening rounds using degenerated primers (see Supplementary Table 1). Primers were based on literature, or designed based on conserved regions in ketosynthase (KS) domains of PKS and adenylation domains (A-domains) of NRPS. Specific PCR for serine activating A-domains was performed as nested PCR, i.e., the PCR products of a general A-domain PCR with degenerated primers were gel purified and used in a subsequent PCR with AnSerin/JS002 as primer pair. The amplified products of approximately 700bp (KS domains), 700bp (A3–A7 region of A-domains), 1000bp (A2–A8 region of A-domains) and 450bp (serine specific nested PCR) were gel purified and cloned into pGEM-T vector (Promega, Madison, WI, United States), transformed into chemical competent E. coli Alpha Silver Select Efficiency (Bioline, Luckenwalde, Germany) and sequenced. Sequences were analyzed using blastx and blastn and closest protein sequences of were analyzed for amino acid specificity using antiSMASH 3.0 (Weber et al., 2015) and submitted to Genbank. The accession numbers for this project are KY671135 – KY671183 (16S rDNA), KY671132 – KY671134 (KS), and KY671184 – KY671199 (A domains).

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for GenElute™ Bacterial Genomic DNA Kit below.

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