Precipitation ethanol

The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

Urea Enzymatic Dialysis Method. This method (16) uses 8 M urea [57-13-6] to gelatinize and facUitate removal of starch and promote extraction of the soluble fiber at mild (50°C) temperatures. EoUowing digestion with heat-stable a-amylase and protease, IDE is isolated by filtration or I DE is obtained after ethanol precipitation. Values for I DE are comparable to those obtained by the methods described eadier, and this method is less time-consuming than are the two AO AC-approved methods. Corrections for protein are required as in the AO AC methods. 

Iodine was determined by an iodometric titration adapted from White and Secor.(3) Instead of the normal Carius combustion, iodide was separated from the samples either by slurrying in 6M NaOH, or by stirring the sample with liquid sodium-potassium (NaK) alloy, followed by dissolving excess NaK in ethanol. Precipitated plutonium hydroxides were filtered. Iodine was determined in the filtrate by bromine oxidation to iodate in an acetate buffer solution, destruction of the excess bromine with formic acid, acidifying with SO, addition of excess KI solution, and titrating the liberated iodine with standard sodium thiosulfate. The precision of the iodine determination is estimated to be about 5% of the measured value, principally due to incomplete extraction of iodine from the sample. 

Mung bean seedlings at different stages of development were treated with 24% (by weight) KOH to extract non-cellulosic polysaccharides as fully as possible. After neutralisation of the extracts, the polysaccharides were isolated by ethanol precipitation, and... 

Crude polysaccharide fraction (GL-2) was prepared from the leaves of P. ginseng by hot water extraction, ethanol precipitation and dialysis, and GL-2 was fractionated by Cetavlon precipitation and weakly acidic polysaccharide fraction (GL-4) was obtained[3]. GL-4IIb2 was purified from GL-4 by DEAE-Sepharose CL-6B as described previousely [3]. In order to remove the color-materials, GL-4IIb2 was further purified by Q-Sepharose (C1 form), and the major fraction, eluted with 0.3 M NaCl, was repurifled by gel filtration on Bio-gel P-30 column to obtain purified active polysaccharide, GL-4IIb2. ... 

Purify conjugate by gel filtration, dialysis or ethanol precipitation EDC = l-ethyl-3-(3-dimethylaminopropyl)carbodiimide HQ. 

Co-precipitate probe and RNA with 1/10th volume 5 M NH4OAc and 2.5 volumes ethanol. Precipitate at —20° for at least 15 min. 

The in vitro transcribed RNAs are phenol-chloroform extracted, ethanol-precipitated, and washed once with 70% ethanol. The RNA pellet is resuspended in 30 1 H20 and loaded to DyeEx 2.0 spin columns (Qiagen) to remove free nucleotides the RNA is then quantified by spectrophotometry. Approximately 20 to 30 pg of RNA is obtained from one reaction. For the initial preparation of transcripts, we would examine the quality and quantity of synthesized RNA by separating them on 1% denaturing agarose gels with total cell RNA preparation as a size maker (28S and 18S ribosomal RNAs... 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

The sample is loaded at a flow-rate of 1 ml/min onto the FPLC column equilibrated with the same MOPS buffer used to resuspend the RNA pellets. The free nucleotides are completely removed with a 5-ml wash with 350 mM NaCl and the RNA is eluted with a 20-ml (350—750 mM NaCl) linear gradient and analyzed by PAGE/urea gel electrophoresis (see later). Up to 2 mg of RNA can be loaded onto and eluted from a 1-ml (of resin) mono Q column without loss of resolution. The homogeneity of RNA in the fractions collected, as seen by gel electrophoresis, should be >90%. The appropriate fractions are pooled and the RNA collected by ethanol precipitation. The RNA pellet is washed twice with 70% ethanol, air-dried, and finally redissolved in DEPC-treated H20. The total recovery after the entire procedure of purification is = 90%. This protocol yields = 800 pmoles of purified 002 mRNA/pmole template DNA. 

Oxidation of the cap structure To 400 fd of 32P-cap-labeled mRNA, 1.6 fd of 100 mMNaI04 is added and left on ice for 2 h, protected from light. The reaction is terminated by addition of 20 fd of 50% glycerol, passed through a G50 spin column and ethanol precipitated. The pelleted... 

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. 

Purify the biotinylated DNA probe by ethanol precipitation, gel filtration, w-butanol extraction, or dialysis as discussed in previous sections. 

Cold composite curve, 23 191 Cold-ethanol precipitation, 22 135-136 Cold exhaust dyeing, 9 176-177 Cold flow improvers, for diesel fuel, 22 427-428... 

Genomic DNA was isolated from each cell hne using standard techniques, which involved sequential washing in PBS and lysis in the presence of proteinase kj 0.4% SDS (Zuccotti and Monk, 1995). Following multiple phenol/chloroform extractions and ethanol precipitations, the amount from each line was... 

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. 

Fpooedme B. Nine grams (dry basis) of ethanol- precipitated and edr-dried graft ocpolymer was mixed with 81 inL of 0.7N sodium hydroxide in a 250-mL Erlenmeyer flask. The flask was heated on a steam bath for 10 min, loosely stoppered, and placed in a 100 C oven for either 1.5 or 3 h. Only about 1 g of volatile material was lost during heating. Ihe cooled reaction mass was methanol-precipitated as described in Procedure A. 

Acid Hydrolysis. Ethanol-precipitated and air-dried graft copolymers were acid-h drolyzed to determine both wei t % nthetic polymer in the graft ocpolymer (% add-on) and the AASO3H content... 

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