A rapid and simple method for DNA preparation fromCandida utilis

A method for the extraction of genomic DNA from the industrial yeastCandida utilis is described. The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should be suitable for Southern blotting and the construction of gene libraries.     

A simple and rapid method for the preparation of apolipoproteins for electrophoresis.

A new method is described for the rapid preparation of apolipoproteins for polyacrylamide gel electrophoresis. It is suitable for all serum lipoproteins including chylomicrons. The procedure involves extraction with diethyl ether in the presence of trichloroacetic acid and sodium deoxycholate. The method gives an improved protein recovery, in particular with chylomicrons. In addition, samples do not require dialysis to remove salts (e.g., KBr) prior to processing; as a consequence, the procedure requires only 1 h. Due to this rapidity and the high yields, the procedure is superior to present methods utilizing ethanol-diethyl ether extraction.     

A rapid method for preparation of nuclear membranes from mammalian cells.

A simple, rapid procedure for disrupting nuclei of mammalian cells has been characterized. This procedure involves treating isolated nuclear suspensions with the natural polyanion heparin in 0.125 m sucrose buffered between pH 7.0 and 8.0. The rate and extent of nuclear lysis are dependent upon the ratio of nuclei concentration to heparin concentration. This procedure avoids use of intense mechanical disruption, enzymatic digestion, and high salt concentrations for achieving optimum lysis of the nuclei. This method can also be used for large-scale nuclear membrane preparations.     

A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles.

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.     

A rapid,simple method for isolating pinocytotic vesicles and plasma membrane of lung

We have found means of isolating pinocytotic vesicles and attached plasma membrane from the low speed (200 × g) supernatant of homogenates of lung. In lung, 5′-nucleotidase is restricted to pinocytotic vesicles and areas of incipient vesicle formation along the plasma membrane. In our method, P_i released from AMP is precipitated as lead phosphate at the subcellular site of 5′-nucleotidase. The resulting increase in density allows collection of pinocytotic vesicles and attached plasma membrane as a pellet after centrifugation through sucrose (d = 1.18) at 250 × g. The final pellet contains long strands of plasma membrane, and the vesicles retain their characteristic morphology including the delicate diaphragm covering their stomata. The entire procedure can be performed in less than 90 min.     

A simple method for preparation of D-rhamnose

A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed. It involves purification of LPS from Pseudomonas syringae pv. phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose. The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.     

A simple rapid immunoassay for S-adenosylhomocysteine in plasma

The measurement of plasma S-adenosylhomocysteine is a more sensitive indicator of the risk for vascular disease than is plasma homocysteine. Because the level of S-adenosylhomocysteine is normally in the nanomolar range, it has been difficult to measure and necessitated the development of complex fluorometric and mass-spectrophotometric methods. We have now adapted an existing immunoassay used for the measurement of homocysteine to the measurement of S-adenosylhomocysteine in plasma. This assay is sensitive down to the level of less than 0.1 pmol, and there is no interference by S-adenosylmethionine. The assay is carried out in microplates, allows the measurement of 12 samples per plate and can easily be carried out in a 4-h period. The method is applicable to plasma samples having S-adenosylhomocysteine concentrations ranging from 10 to 150 nM without dilution. The mean value for 16 normal subjects by this method was 18.9±1.4 nM (S.E.M.), compared with 17.8±1.4 nM obtained by a previously described method using two high-performance liquid chromatography columns with fluorescence derivatization. Mean values for seven cirrhotic patients were 46.5±3.3 nM by this new method compared with 44.6±5.3 by the former method. The ease and speed of this method should allow the widespread measurement of this important metabolite in laboratories without access to sophisticated equipment.     

一种简易的小牛血清制备方法

小牛血清是细胞组织培养中常用的培养基成份。介绍一种简易、快捷的小牛血清制备方法,经该方法制备的血清质量稳定,其在实际应用中效果良好。     

A simple and rapid preparation of alditol acetates for monosaccharide analysis

A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.     

A rapid method for preparation of bacterial plasmids

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.