Total nucleic acid extraction - NucleoMag® DNA/RNA Water Kit (Macherey-Nagel)

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Total nucleic acid extraction from wastewater using NucleoMag® DNA/RNA Water Kit (Macherey-Nagel)

1. Clean the working area and all equipment: wipe down with 10% bleach and let dry. Wipe down with 70% ethanol and let dry. Then, wipe down using RNase AWAY and let dry.

2. Prepare the 50% ethanol solution (it must be fresh!)

3. Prepare the 6 purification plates:

   • Wash 1 plate : Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.

   • Wash 2 plate (same as plate Wash 1): Add 100 μl of 50% ethanol and 900 μl of wash buffer (WBA) to each well required for purification.

   • Ethanol Wash plate : Add 450 μl of 50% ethanol to each well required for purification.

   • Elution plate : Add 100 μl of 25 mM Tris-HCl (pH 8.0) to each well required for purification.

   • Tip plate : Place KingFisher 96-tip comb into an empty KingFisher 96-well plate. While opening the 96-tip comb plate, pay attention to not touch the tips.

   • Lysis and Bind plate : Add 35 μL of Resin to each well required for purification (v ortex the bottle at max speed before use) . Add 50 μl of Alkaline Protease Solution custom (APA) to each well required for purification. Add 250 μl of cell lysis solution (CLD) to each well required for purification. Add 400 μl of Isopropanol (100%) to each well required for purification.