ABSTRACT
Water bath or thermal block Centrifuges (must be suitable for 2 mL test tubes and for 50 mL test tubes) Vortex Thermalcycler Transilluminator UV-VIS spectrophotometer Complete apparatus for horizontal electrophoresis (including casting trays, electrophoretic
chamber, and power supply) ABI 310 or 3100 sequencer (if not available, the capillary electrophoresis of digests can be
carried out by an external subcontractor) Microwave
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 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 0.5M 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 6X (Promega, Fermentas, or similar) Molecular weight 100 bp (Fermentas or similar) Wizard® PCR cleanup system (Promega) Alu I restriction enzyme (Promega, Fermentas, or other) Formamide Internal size standard (GS1000ROX; Applied Biosystems)
The application of the 16S rDNA gene T-RFLP method to sediment samples involves the following steps:
1. DNA extraction and purication (with the aim of extracting genomic DNA from benthic microrganisms, which must be of sufcient purity to carry out subsequent PCR-based analyses).
