Ethanol precipitation step

El 4. What is the purpose of the ethanol precipitation step in the preparation of plasmids ... 

If desired, a TaKaRa Redochip can be used instead of the DE81 ion-exchange paper. In this case, the elution, extraction, and ethanol precipitation steps (steps 8-12 above) can be omitted. 

First, the RNA must be collected from the transcription mixture by precipitation. We use ammonium acetate in the ethanol precipitation step because this is the quickest method. Coprecipitated NH ions, which interfere with further enzymatic reactions, are removed in the next purification step. The RNA pellet is dissolved in an appropriate loading buffer for denaturing PAGE (7 M urea, 50 mM EDTA), incubated for 5 min at 65 °C, and then immediately loaded on the gel. The gel should be preequilibrated and preheated by preelectrophoresis for 15 min. 

In the ethanol precipitation step, the use of sodium acetate instead of ammonium acetate should be avoided, because trace sodium salt may interfere with the translation reaction. 

Problems are sometimes encountered in contamination of the DNA with acrylamide monomers or other impurities from the gel matrix. Such contamination is usually apparent from the formation of an excessive turbidity during the ethanol-precipitation step. Pure DNA preparations in the microgram amounts eluted rarely give visible turbidity. A convenient purification procedure is to absorb the DNA onto a small (1 ml) column of DEAE-cellulose (DE-52, Whatman) in a low ionic strength buffer. Non-ionic im-... 

Residual salt from ethanol-precipitation step suppressing reaction of T with hydrazine... 

Reduction in viral infectivity occurs by inactivation of virus or by removal of virus particles. During removal steps, virus is not inactivated but is separated from the protein of interest using methods such as precipitation, chromatography, or filtration. For example, during an ethanol precipitation step, ethanol is added to a suspension to precipitate unwanted contaminating proteins and viruses. The ethanol-containing suspension is then centrifuged so that the contaminants in the precipitated paste fraction can be separated from the product in the effluent fraction. 

A good stopping point in the end-labeling reaction is the ethanol precipitation step after the phenol/chloroform extraction. The RNA precipitation can be left at -20°C from an hour to overnight. 

The addition of glycogen to the ethanol precipitation step after labeling and gel purification increases the recovery of the precipitated RNA. After ethanol precipitation, the white RNA pellet is easily displaced from the bottom of the tube if the tube is jostled or allowed to stand. Remove the supernatant immediately after the microfuge centrifugation is completed. Take special care with the second pellet from the ethanol wash—it seems to stick less well to the tube and often floats loose. If this happens, use a pipet tip and carefully remove the supernatant without sucking up the pellet—do not use the aspirator. 

NH4)2S04 from ethanol or acetone powders (Ochoa, 1955). An additional zinc-ethanol precipitation step yielded apparently pure preparations (Wolfe and Neiland, 1956). More recently, large-scale purification of pig-heart MDase was achieved by fractionation on CM-cellulose, followed by further purification on Sephadex G-lOO (Glatthaar et al., 1974). Mitochondrial MDase from acetone powders was also obtained by precipitation with (NH4)2S04, followed by chromatography on Bio-Rex 70 and DEAE-cellulose (Gregory et al., 1971). 

If protein contamination is a problem further phenol/chloroform extractions (steps 4-6) followed by ethanol precipitation (steps 7 and 8) should be carried out. 

Tris and boric acid and 10 mM EDTA, 80 V, 1 hr). The presence of 28S and 18S rRNA banding in a ratio of 2 1 in both RNAs would indicate removal of nuclease from the RNA. If the 28S/18S ratio is less than 2 1 in the incubated RNA, carry out another cycle of the GH-ethanol precipitation step further to remove the residual nuclease activity. 

TCA precipitation may be replaced by DE81 filter-binding assay (20). Actually, this entire step may be omitted m routine expenments and replaced by simple counting of an aliquot of the final reaction product (resuspended e.g, in 100 xL). Note, however, that a fraction of the unincorporated radionucleotides may not be eliminated after the two ethanol-precipitation steps. Therefore, a final radioactivity count may overestimate the actual amount of probe. 

Purify the labeled probe by ethanol precipitation according to steps 4-7 of the protocol previously described for nick translation. 

The diamine-modified DNA may be isolated from excess reactants by ethanol precipitation according to steps 4-7 of the protocol described previously for nick translation (Section 1, this chapter). Alternatively, dialysis or gel filtration may be done to remove excess reactants. 

In the first step, 2.2 g of the ethanol-precipitated solid were dissolved in 55 mL of 0.05M sodium phosphate buffer at pH 7.3, the undissolved residue was removed by centrifugation, and the supernatant was added to a 50-mm i.d., 180-mm long DEAE-cellulose column held at room temperature and eluted with 3.33 mL/min of the same buffer. Since the isoelectric point of the desired xylanase was above the buffer pH, it passed through the column without being retarded, and contaminating protein was removed. 

Phenol- and chloroform-extract and ethanol precipitate the recovered RNA (see Subheading 3.7). The RNA is reverse transcribed, purified, and precipitated as detailed in Subheadings 3.6 (25) and 3.7, and then RNA is used for the nitrocellulose filter selection step and cocaine displacement of nAChR-bound RNA molecules (Fig. 2). 

To an appredable extent the solubility of nitroglycerine in ethyl alcohol depends on the temperature of the solvent and its water content. In the cold a limited quantity of nitroglycerine is dissolved in absolute alcohol, whereas at temperatures of about 50°C it mixes with absolute or 96% alcohol in all proportions. The solubility of nitroglycerine decreases as water is added to the ethanol. In consequence, when water is poured into an alcoholic solution, nitroglycerine is precipitated step by step. This precipitation becomes very considerable after the alcohol has been diluted from 50 to 25% concentration. 

Purify the reaction products by using a PCR purification kit or by ethanol precipitation (see Section 3.3.2 steps 5-10), digest with appropriate restriction endonucleases, and ligate into the cloning vector. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

Our laboratory has found that the inclusion of an additional purification step involving ethanol precipitation from an aqueous solution made... 

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