A simple, efficient method to create a cDNA library.

The simplified "All In One Tube" protocol for constructing a cDNA library combines the advantages of the "Classic method" and the Okayama-Berg method while overcoming some of their drawbacks. In this method, adding adapters, linkers or enzyme digestion steps are no longer necessary after cDNA synthesis, thus making it quicker and especially useful when dealing with small samples of mRNA.     

A simple and efficient synthesis of puromycin, 2,2'-anhydro-pyrimidine nucleosides, cytidines and 2',3'-anhydroadenosine from 3',5'-O-sulfinyl xylo-nucleosides

Synthesis of antibiotics, puromycin and 3'-amino-3'-deoxy-N6,N6-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3',5'-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-alpha-carbamate 7. In a similar manner 2,2'-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2',3'-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

A simple and efficient method to remove ribonuclease contamination from pancreatic deoxyribonuclease preparations

An affinity chromatography method which efficiently removes the contaminating RNase activity from commercial pancreatic DNase preparations is described. The new method is compared with the iodoacetate treatment previously used for the preferential inactivation of RNase.     

A simple method to vary soil heterogeneity in three dimensions in experimental mesocosms

Soil heterogeneity affects terrestrial plant communities both directly and indirectly. In nature, the exploration of the role of heterogeneity is made difficult because any co-varying factors (nutrients, soil depth, etc.) render it problematic to clearly link cause and effect. Attributing changes specifically to heterogeneity is facilitated if heterogeneity is varied in a controlled manner and other possible confounding factors are kept constant. The experiments conducted in such a way have up till now only considered heterogeneity in two dimensions, horizontally or vertically. In this methodological study, we present a novel technique that enables researchers to vary both qualitative and configurational heterogeneity in three dimensions by building up the soil cell by cell in experimental mesocosms. We illustrate the technique with an experiment where we test the effect of cell size (i.e. configurational heterogeneity) on the performance of grassland species that vary in nutrient preference (high N and low N species). Cell size did not affect aboveground biomass but modified species richness, both at the mesocosm and the patch scale, with most species being found when cells were small yet distinct (cell size 12 cm). High N species had significantly greater aboveground biomass and species richness than low N species, both on nutrient rich and nutrient poor cells. Remarkably, those differences disappeared when plants grew on the mesocosms with cell size close to zero. By allowing greater complexity in the design of experimental mesocosms, the 3-D approach can improve understanding of the interplay between soil heterogeneity and plant and ecosystem functioning.     

A simple and efficient method for predicting protein-protein interaction sites

Computational methods for predicting protein-protein interaction sites based on structural data are characterized by an accuracy between 70 and 80%. Some experimental studies indicate that only a fraction of the residues, forming clusters in the center of the interaction site, are energetically important for binding. In addition, the analysis of amino acid composition has shown that residues located in the center of the interaction site can be better discriminated from the residues in other parts of the protein surface. In the present study, we implement a simple method to predict interaction site residues exploiting this fact and show that it achieves a very competitive performance compared to other methods using the same dataset and criteria for performance evaluation (success rate of 82.1%).     

A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.     

A new method for the mapping of 5' ends of RNAs

In this article, we describe a new procedure to map 5′ ends of RNAs. The procedure consists in the use of specific RNase H digestion of a hybrid formed by the RNA and a complementary DNA oligonucleotide. Northern blot hybridization of the resulting RNA fragment allows an accurate measurement of its length. Although we generally use this procedure as a control of previously performed primer extension analyses, the absence of nonspecific bands, which often occur in primer extensions on RNA templates with extended secondary structures, suggests that our method may be preferable when these difficult templates are analyzed.     

A simple and efficient method for preparing and dispensing anaerobic media

Summary An Oxford pipettor (model S-A) was used for simultaneously preparing and dispensing anaerobic media. Media prepared by this method have been successfully used for cultivation of extremely thermophilic bacteria.     

A simple and efficient method of labelling RBCs with 99mTc for spleen imaging

A new method of labelling human RBCs with ^99mTc by the use of Sn-α-d-glucose 1-phosphate (GP) is presented. It was tested for spleen imaging in 16 normal volunteers. The labelling was carried out during the heating (30 min at 49.5 °C) to damage the cells and cooling (10 min at R.T.) steps. The labelling efficiency was 95.5 ± 1.5% with Sn-GP and was better than Sn-PYP (61.5 ± 25.0%). The radioactivity retention of RBCs was > 96% after 6 washings. The spleen was delineated very well in all the subjects. The spleen activity reached a plateau at 20 min post-injection. Spleen-to-liver and spleen-to-cardiac blood pool concentration ratios were high (10.4 ± 2.2 and 10.1 ± 3.2, respectively) calculated at 30 min. The method is simple, rapid and efficient.     

A simple and efficient method for raising steroid antibodies in rabbits

The production of estradiol antibodies by two immunization procedures was monitored by means of an enzyme-immunoassay. Procedure A consisted of three intramuscular injections given at two-week intervals, followed by five intravenous booster injections and procedure B consisted of multiple intradermal injections given once. Procedure A gave much higher antibody titer. In both procedures the sensitivities of assays using the antisera increased initially and reached a plateau after three to four months of immunization. Consistent changes in specificities were observed. A shortened procedure A is proposed as a simple and efficient procedure for raising steroid antibodies in rabbits.     

A simple and efficient cryopreservation method for primate embryonic stem cells

Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slow-rate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

A simple and efficient microassay method for titration of interferon.

A simple and efficient microassay method for the titration of interferon was developed by the use of microtest plates for handling a large number of samples. L929 cells pretreated with interferon were infected with vesicular stomatitis virus (VSV) and cultured in the presence of 3H-uridine. The activity was expressed by the reduction of extracellular radioactive RNA released after destruction of the infected cells, which was measured in terms of the radioactivity incorporated into cold TCA-insoluble materials in the culture fluid. The interferon titer determined by this method was in the same order as that by the plaque reduction method. The activity by this method was parallel to, but lower than that expressed by the yield reduction of infectious viruses. This method requires only 0.025 ml of each test sample with higher than 1 NIH ref. unit/ml to detect its interferon activity and takes 2 to 3 days for assaying hundreds of samples.