Combisart 6 branch manifold

Recombinant plasmid DNAs pBELX and pNORM1, used as qPCR standards, were extracted using the “Wizard^® Plus SV Minipreps DNA Purification System” (Promega) according to the recommendations provided by the manufacturer. The plasmids were linearized by BamHI (Promega) before being purified with the “QIAquick PCR DNA purification kit” (Qiagen). Total environmental DNAs were extracted using the “PowerWater DNA Isolation Kit” (MO BIO laboratories Inc). Briefly, 50 mg of sediments or SMs were thawed before being dispersed in 100 mL of non-pyrogenic sterile water (Aqua B-Braun) by vortexing for 30 s followed by 15 min stirring at 160 rpm and 25°C. These sediment/SM suspensions (or 100 mL of sample for raw waters) were filtered on polycarbonate filters (Whatman Nuclepore filter, pore size 0.22 μm, diameter 47 mm) using a filtration apparatus (Combisart 6-branch Manifold, Sartorius). Total DNAs were directly extracted from the filters according to the recommendations provided by the manufacturer and were eluted from silica columns with 100 μL of PCR grade water (RNase-Free Water, Qiagen). Plasmid and total DNA concentration and purity were estimated by spectrophotometry according to standard procedures, and all DNAs were stored at -20°C until use.