ABSTRACT
Centrifuges (must be suitable for 2 mL and 50 mL test tubes) Vortex Thermalcycler Transilluminator UV-VIS spectrophotometer Complete apparatus for horizontal electrophoresis (including casting trays, electrophoretic
chamber, and power supply) ABI (Applied Biosystems) 310 or 3100 sequencer (if not available, the capillary electrophore-
sis of puried PCR products can be carried out by an external subcontractor)
Wash Solution 1: 50 mM Tris-HCl, pH 8.3; 200 mM NaCl; 5 mM Na2EDTA; 0.05% Triton X-100
Wash Solution 2: 50 mM Tris-HCl, pH 8.3; 200 mM NaCl; 5 mM Na2EDTA Wash Solution 3: 10 mM Tris-HCl, pH 8.3; 0.1 mM Na2EDTA Ultrapure water: Reagent-grade water, previously autoclaved, ltered (through 0.2 µm) and
stored into sterile 2 mL Eppendorf tubes at 4°C or −20°C UltraClean™ Soil DNA isolation kit (MoBio, catalog #12800-100) MasterTaq® kit (Eppendorf AG, Germany) Agarose TBE (Tris-Borate-EDTA)10× (can be purchased from BIORAD or similar or, alternatively,
prepared as follows [for 1 L]: 108 g TRIS Base; 55 g Boric Acid; 40 mL EDTA 0.5 M, pH 8; reagent-grade water to volume)
TBE 1×: Dilute TBE 10× (100 mL TBE 10× + 900 mL of reagent-grade water) Ethidium bromide (10 mg mL−1 in ultrapure water) Loading dye 6× (Promega, Fermentas, or similar) Molecular weight 100 bp (Fermentas or similar) Wizard® PCR cleanup system (Promega) Formamide Internal size standard (GS2500-ROX; Applied Biosystems)
The application of the ARISA method to sediment samples involve the following steps:
1. DNA extraction and purication (with the aim of extracting genomic DNA from benthic microrganisms, which must be of sufcient purity for subsequent PCR-based analyses).
