Cooling water biofilm
DNA extraction is done using Xploregen kit. Add 60 ul of solution C1 and invert several times or vortex briefly [Note: Solution C1 maybe added to the power bead tube before adding sample] Vortex at max speed for 10 mins. [Note: If using the 24-place vortex adapter for more than 12 preps, increase the vortex time by 5-10 mins] Centrifuge tube at 10,000 x g for 30s Transfer the supernatant to a clean 2ml collection tube. [Note: expect between 400 to 500 ul of supernatant. Supernatant may still contain some particles.] Add 250 ul of soln. C2 and vortex for 5 sec. Incubate at 4 degree C for 5 mins.  Centrifuge the tube for 1 min at 10,000 X g Avoiding the palette, transfer up to 600 ul of supernatant to a clean 2ml collection tube. Add 200 ul of soln. C3 and vortex briefly. Incubate at 4 degree C for 5 mins. [Note: You can skip 5 min incubation. However, if you have already validated the extraction with this incubation, we recommend you to retain the step.] Centrifuge the tube for 1 min at 10,000 X g.  Avoiding the palette, transfer up to 600 ul of supernatant to a clean 2ml collection tube.  Shake to mix soln. C4 and add 1200ul to the supernatant. Vortex for 5 s. Load 675ul onto an MB Spin Column and centrifuge at 10,000 X g for 1 min. Discard flow through.  Repeat the above step twice, until all of the samples have been processed.  Add 500 ul of the solution C5. Centrifuge for 30s at 10,000 X g. Discard the flow through. Centrifuge again for 1 min at 10,000 X g. Carefully place the MB spin column into a clean 2ml collection tube. Avoid splashing any solution C5 onto the column.  Add 100ul of the soln. C6 to the center of the white filter membrane. Alternatively, you can use sterile DNA-free PCR grade water for this step. Centrifuge at room temperature for 30s at 10,000 X g. Discard the MB spin column. The DNA is now ready for downstream application. [Note: Solution C6 is 10 mMTris-HCl, pH 8.5. We recommend storing DNA frozen (-20 degree C- -80 degree C) as soln. C6 does not contain EDTA.]PCR Amplification of 16s Region:Composition of TAQ Master MIX:1) High-Fidelity DNA Polymerase 2) 0.5mM dNTPs3) 3.2mM MgCl24) PCR Enzyme BufferPrimer Details: 16sF:- 5 AGAGTTTGATGMTGGCTCAG316sR:- 5 TTACCGCGGCMGCSGGCAC3Conditions Used:40ng of Extracted DNA is used for amplification along with 10pM of each primerInitial Denaturation - 95 degree C25 Cycles of the following condition,Denaturation @ 95 deg C for 15 sec, Annealing @ 60 deg C for 15 sec, Elongation @ 72 deg C for 2 minsFinal Extension at 72 deg C for 10 mins and Hold at 4 deg C.DNA QC :Extracted DNA from the samples were subjected to Nano Drop and GEL Check before being taken for PCR amplification:The Nano Drop readings of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs qualityPCR Ampliqon QC:The amplified 16s PCR Product is purified and subjected to GEL Check and Nanodrop QCThe Nano Drop readings of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs qualityOverview of Sequencing Protocol:The amplicons from each sample were purified with Ampure beads to remove unused primers and an additional 8 cycles of PCR was performed using Illumina barcoded adapters to prepare the sequencing libraries. Libraries were purified using Ampure beads and quantitated using Qubit dsDNA High Sensitivity assay kit. Sequencing was performed using Illumina Miseq with 2x300PE v3-v4 sequencing kit.