[[Microbial Ecology Methods]]

buffers_and_reagents:dapi - created

Boris Wawrik — 2008-07-31T15:04:26-04:00

4'-6-Diamidino-2-phenylindole (DAPI) stains double stranded DNA and emits blue light under UV excitation. It is typically used for counting bacterial cells in natural samples. A typical stock of DAPI is made as 5 mg / ml in DI, DMSO, or methanol. I typically use DI water. A working concentration of DAPI is 5 ug / ml, which means that this stock is 1000x i.e. to stain cells in one ml of sample, add 1 ul of DAPI stock solution.

start

Boris Wawrik — 2008-07-31T14:54:20-04:00

This site contains some common methods/protocols in microbial ecology. The material originates from many different places and some of it may be taken directly from other sources. I try to give credit where I can, but don't always explicitly state my source. So please use caution. The information is not intended for distribution and was written as a document for internal use only.

genomics:preparation_of_dialysis_bags

2008-03-31T16:51:07-04:00

This procedure is used to prepare dialysis bags for electroelution of partially digested high molecular weight (HMW) DNA from agar strips. HMW DNA is then cloned into a BAC vector. Cleaning solution 1 mM EDTA pH 8.0 2% NaHCO3 Cleaning procedure * Place dialysis bag into cleaning solution * Heat at 90º C for 10 minutes * Remove dialysis bags and place in DI * Boil for 10 minutes * Wash several times in fresh DI * Store in 50 % Ethanol at 4º C * Wash tubing in sterile DI immed…

phytoplankton:phytoplankton_glassware

2008-03-31T16:45:35-04:00

Phytoplankton glassware, containers, tubing and reagents should be carefully selected to avoid toxic compounds. Generally borosilicate glassware and tissue culture-grade polycarbonate or polystyrene plasticware are recommended. Teflon-lined caps are desirable for screw-top glass test tubes. Black caps should be autoclaved several times in changes of seawater. This helps to reduce teh leakage of toxic phenolics when heated.

dna_extraction:dissolved_dna_using_hoechst_33258 - created

Boris Wawrik — 2008-03-31T11:21:06-04:00

The is a fluorometric method for quantifying DNA. It tends to be useful when organics contaminate DNA extracted from an environmental sample. In this situation a measurement of A260/A280 does not give accurate DNA measurements. The method is based upon the interaction of the bisbenzimide fluorochrome Hoechst 33258 with the major groove of AT-rich portions of double stranded DNA. Standard curves are generated using calf thymus DNA or salmon sperm DNA

dna_extraction:dna_extraction_from_seawater_-_john_paul

2008-03-31T10:24:29-04:00

Before starting: * Makes sure that screw cap microcentrifuge tubes are available (sterile). I prefer 2.0 ml but 1.5 ml will suffice. * Solutions, freshly made and autoclaved: * TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) * STE (100 mM NaCl, 10 mM Tris-HCl, 1mM EDTA, pH 8.0) * 3 M Sodium Acetate pH 7.0 * TE saturated Phenol * Chloroform:IAA 24:1 * 20 % SDS

phytoplankton:sn_medium

2008-03-31T00:01:14-04:00

Filter 750 mL seawater through a 0.22 uM filter and add 250 mL distilled H2O. Autoclave in a teflon lined bottle then add the following compounds aseptically: Quantity Compound Stock Solution 2.5 mL NaNO3 300.0 g/L dH2O 2.6 mL K2HPO4 (anhydrous) 6.1 g/L dH2O 5.6 mL Na2EDTA.2H2O 1.0 g/L dH2O 2.6 mL Na2CO3 4.0 g/L dH2O 1.0 mL Vitamin B12 (Cyanocobalamin) 1.0 mg/L dH2O 1.0 mL Cyano Trace Metal Solution (see recipe below) CCMP modifies Waterbury's SN Me…

phytoplankton:media_and_cultures

2008-03-27T09:05:39-04:00

Medium and Cultures A good place for more information about cultures, ordering cultures, and medium formulations can be found on the Provasoli-Guillard National Center for Culture of Marine Phytoplankton - CCMP (USA) website: