EDTA Extraction Protocol

The extraction should be performed in 250 mL pre-cleaned borosilicate glass, polypropylene or PTFE bottles using an end-over-end shaker. All laboratory glassware should be cleaned with HCl, rinsed with distilled water, cleaned with 0.05 mol L EDTA and rinsed again with distilled water. 

Extraction should be batch-wise, followed by centrifugation according to the following procedure. A 5 g soil sample should be transferred to an extraction bottle in which 50 mL of 0.05 mol L EDTA is added. The mixture obtained should be shaken on an end-over-end shaker operating at 30 rpm for 1 h in a room at 20 °C. 

The temperature of the room should be measured at the beginning and at the end of the extraction, as well as the temperature of the extracting solution in the bottle at the end of the shaking period. The extracts should be immediately filtered through a filter paper (porosity 0.4-1.1 pm capable of retaining particles of 2.7 pm size) previously rinsed with 0.05 mol L EDTA followed by distilled water. The filtrates should be collected in polyethylene bottles. Blank extractions i.e. without soil) should be carried out for each set of analyses using the same reagents as described above. 

The sample for analysis should be taken as it is. Before a bottle is opened it should be manually shaken for 5 min to rehomogenize the content. The results should be corrected for dry mass this correction must be performed on a separate portion of 1 g taken at the same time from the same bottle by drying in an oven at 105 2°C for 2-3 h until constant mass is attained (successive weighings should not differ by more than 1 mg). 

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In order to improve comparability between results of extraction protocols for soil plant-available PTMs obtained by different laboratories, the Measurement and Testing Programme (formerly BCR) of the European Commission has recently proposed two harmonised procedures for extractable Cd, Cu, Cr, Ni, Pb and Zn in soils on the basis of 0.05 M EDTA (pH 7.0) and 0.43 M acetic acid solutions (Rauret et al, 2001). Moreover, a suitable reference material (BCR-700 organic-rich soil) for these extractions was prepared, which enable the quality of the measurements to be controlled. 

A DNA extraction protocol that has proved useful for most ancient tissues is a modification of the protocol initially published by Blin and Stafford.20 Approximately 0.1 g of small pieces of soft tissue is added to 5 ml of extraction buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 10 mg/ml dithiothreitol (DTT), and 0.5 mg/ml proteinase K. Incubation at 37° with gentle agitation overnight will allow most or all of the tissue to go into solution. An equal volume of phenol, equilibrated with 1 M Tris-HCl (pH 8.0), is added. When the phenol is being equilibrated, care should be taken to use uncontaminated Tris buffer and to measure the pH only on aliquots that are removed from the water phase and then discarded. Two phenol extractions and one chloroform extraction are performed, and the water phase is concentrated and purified on a Centricon 30 microconcentrator (Amicon, Danvers, MA). The reten-tate can be stored frozen, preferably in a few aliquots. In all cases solutions should be manipulated with DNA-free positive displacement pipettes. 

For the homogeneity studies, the extractants (0.05 mol L EDTA, 0.43 mol L" acetic acid and 0.005 mol L DTPA) were prepared as laid out in the certification reports [15, 17], The trace element contents (Cd, Cr, Cu, Ni, Pb and Zn) in the extracts were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) for the CRMs 483/484, flame atomic absorption spectrometry (FAAS) or electrothermal atomic absorption spectrometry with Zeeman background correction (ZETAAS) for the CRM 600. In the case of the CRM 483, little analytical difficulty was experienced as illustrated by the good agreement obtained between the within-bottle and between-bottle CVs for the CRM 484, lower extractable contents, closer to the detection limits and consequent poorer analytical precision was observed in particular for Cr (EDTA extractable contents), Cd and Pb (acetic acid extractable contents). No particular difficulties were experienced for the CRM 600. On the basis of these results, the materials were considered to be homogeneous at a level of 5 g for EDTA- and acetic acid-extractable contents and 10 g for DTPA-extractable contents (as specified in the extraction protocols). 

The project was started in 1987 by a consultation of Emopean experts examining the possibility of harmonizing single and/or sequential extraction schemes for soil and sediment analysis [60]. This inquiry was followed by the design of single extraction schemes (EDTA, acetic acid and ammonium acetate) and a sequential extraction protocol which were proposed to a group of ca. 30 laboratories for possible harmonization in 1989 [193]. Interlaboratory studies (two on soils and two on sediment) were carried out between 1989 and 1993 [194-196] and were followed by certification campaigns conducted between 1994 and 1996 [197-199]. 

In the fields of environmental and exploration geochemistry, considerable use has been made of sequential extraction procedures in order to determine the bioavailability and geomobUity of trace metals (including V, Cr, As, Se, Cd and Pb). More recently, sequential extractions have been used to elucidate information relating to the speciation of metals in soils and sediments. The extraction protocol developed by Tessier " has traditionally been used (with modifications) and involves the sequential use of a variable cocktail of extractants including acetic acid, sodium acetate, magnesiumchloride, EDTA, acidified hydroxylamine hydrochloride, oxalic acid, sodium... 

Classical approaches to plant DNA isolation aim to produce large quantities of highly purified DNA. However, smaller quantities of crudely extracted plant DNA are often acceptable for PCR analysis. Another efficient method for preparation of plant DNA for PCR is a single-step protocol that involves heating a small amount of plant tissue in a simple solution. Several factors influence nucleic acid release from tissue salt, EDTA, pH, incubation time and temperature. These factors must be optimized for different sample substrates. EDTA in the sample solution binds the Mg + cofactor required by the Taq polymerase in the PCR, so the EDTA concentration in the solution, or the Mg + concentration in the PCR, must be carefully optimized. 

Since size heterogeneity of LPS molecules can exist within a single organism, a extraction method applied to both S-LPS and R-LPS extractions has been developed (Darveau and Hancock, 1983). In this method, SDS and EDTA are applied to precipitate contaminants, such as peptidoglycan and proteins. The following is the brief protocol of this method ... 

Bone samples can also be extracted by the above protocol, with yields similar to more specialized protocols. However, the most successful protocols involve the chelation of the hydroxyapatite by 0.5 M EDTA for prolonged periods of time.3 The remaining decalcified tissue is made up of collagen and contains the trapped DNA. The near future will undoubtedly... 

The other popular sequential extraction procedure is the protocol proposed by the Community Bureau of Reference, Commission of the European Community (known as the BCR protocol). The method was proposed on the basis of interlaboratory smdies undertaken in order to harmonize conditions for soil and sediment sample analysis. Based on the research data, in 1992 it was stated that application of EDTA or acetic acid solution is appropriate and sufficient for elimination of the bioaccessible fi action of metals from soil samples [62]. In the case of other samples, best results were achieved after application of a three-stage procedure with the following extractants ... 

Since both the temperature of extraction and the vigour of the mechanical shaking could be expected to affect the amount of metal extracted, limits for both of these parameters were prescribed in the detailed protocol for extraction and analysis issued to the participating laboratories along with the solid sample [193]. As a check on each laboratory s calibration, reference solutions containing the trace metals of concern (Cd, Cr, Cu, Ni, Pb and Zn) were prepared. Three types of single extractant were used EDTA (0.05 mol L" ), acetic acid (0.43 mol L ) and ammonium acetate (1 mol L ). 

The initial PIA purification method was developed by Mack et al. (3). These authors used a different, two-step chromatography protocol involving size-exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose, and S-Sepharose. A similar purification method has been described recently to isolate a PIA-related polysaccharide polymer in E. coli (7). Briefly, E. coli cells were incubated in 50 raM Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and 0.1 M EDTA at room temperature for 2 h. Phenol/chloroform extraction steps were performed to separate protein and debris contamination from the polysaccharide. Samples were concentrated by ultrafiltration devices (10,000 MW cut off) and fractionated on a fast protein liquid chromatography (FPLC) system with a Sephacryl S-2000 column (equilibration and elution buffer 0.1 MPBS, pH 7.4). 

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