Genomic DNA MiniSpin: Bacteria

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

The identity of the selected isolate was confirmed based on 16S rRNA gene sequence analysis. Genomic DNA was isolated from the bacterial sample using Chromous bacterial genomic DNA isolation kit. The universal primers of 16S rDNA fragments, 27F and 1492R, were used to amplify the 16S rDNA. The sequences of primers were as follows: (27F) AGAGTTTGATCMTGGCTCAG and (1492R) TACGGYTACCTTGTTACGACTT (Chromous Biotech, Bangalore). A phylogenetic tree was constructed using partial 16S rRNA gene sequences of the isolate and the other sequences, closely related with the reference strain, obtained from NCBI database. Clustal Omega was used for multiple sequence alignment of sequences. Neighbor joining tree was constructed with complete deletion using bootstrapping at 10,000 bootstraps trials with Kimura-2 parameter using MEGA 6.0 software (Das and Tiwary 2013). The isolate P4 was finally identified as P. otitidis. The sequence of the 16S rRNA gene of the strain P4 is available in NCBI under the GenBank accession number KP877124.

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Manufacturer protocol

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