A simple,efficient, standard-dilution method for cloning hybridomas

Summary A simple standard-dilution method which obviates routine use of viable cell counts, feeder layers and time-consuming scale-up procedures is described. This method can be used to clone monoclonal antibody-producing hybridomas 9–14 days post-fusion. Each cloning cycle takes 5 min. Approximately 60% of the positive, monoclonal antibody-producing hybridomas were successfully cloned and established.     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101／pHQ9,EHA101／pHQ10,EHA101／pHQT3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤／g愈伤外植体,抗性愈伤的分化频率平均高达85％。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3：1。该系统的建立将有助于应用T—DNA标签法和基因打靶法进行水稻功能基因组的研究。     

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 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.     

A simple,efficient method for purification of degraded PCR primers

Summary A simple, efficient method was used to purify PCR primers which had degraded during storage at-20°C/-9°C. The primers were eiectrophoresed on 3% (w/v) agarose gel, the main band was electroeluted via a trough cut in the gel. The primers were recovered by isobutanol extraction followed by ethanol precipitation. The yield was 20–40% and the A₂₆₀/A₂₈₀ ratio was greater than 1.8. The purification resulted in good amplification.     

A simple and efficient method for chemical mutagenesis of DNA.

A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase.     

A simple and efficient method for the preparation of GDP-fucose.

A crude extract from hog submaxillary gland was found to synthesize guanosine diphosphate (GDP)-fucose, when incubated with fucose, ATP and GTP, the two enzymatic steps (fucose

fucose-1-phosphate

GDP-fucose) proceeding without noticeable side reactions. Column chromatography on Dowex 1 (HCO₃^?), gel filtration on Sephadex G-15 and preparative paper chromatography gave pure GDP-fucose in an overall yield of 81%. The sugar nucleotide was shown to be active as a glycosyl donor in fucose-incorporating systems.     

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 for isolating trichomes for downstream analyses

Arabidopsis trichomes are an excellent cell type to address many questions in plant biology including the control of cell shape, endoreplication, and cell expansion. Because trichomes comprise such a small percentage of the cells of a leaf, biochemical analyses of trichomes are limited. To overcome this limitation, we developed a method for removing trichomes from the leaf surface. Our method allows the isolation of intact trichomes for use in downstream applications such as cell wall analysis, immunolocalization of trichome proteins, analysis of DNA content, and proteomics. Also, this method will facilitate the isolation of trichomes from practically any plant species.     

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, 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 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 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.