一种简便、快捷的胰蛋白酶抑制剂基因的分离与克隆方法

从 3个豇豆品种幼嫩叶片中分离出核基因组 DNA,参照已知的几种 Bowman-Birk型胰蛋白酶抑制剂基因序列 ,设计合成了 2 7bp,且含有 Bam H I位点的寡核苷酸引物 ,分别以 3种豇豆核基因组 DNA为模板 ,PCR扩增 ,均得到长度约为 3 40 bp的 DNA片段。产物 DNA片段经 DNA序列分析 ,结果表明三者的碱基序列相同 ,与报道的胰蛋白酶抑制剂基因相比 ,同源性为 1 0 0 %和 99.7%。     

一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

A rapid method for preparing insect microsomes

The 1000 g supernatant of a tissue homogenate is layered on top of a small (less than 5 ml) sucrose density gradient and centrifuged for 20 min at very high centrifugal forces in a vertical rotor. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centrifugation method. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.     

A simple method for cloning leishmanial promastigotes

A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3 X 10(3) promastigotes per ml. Hanging-drop preparations made from 0.2-0.4 microliter volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22 degrees C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species of Leishmania, with up to 100 percent of the cloned organisms growing.     

A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.     

A simple and rapid method for purifying staphylococcal exfoliative toxin A

A rapid and efficient method of purification of staphylococcal exfoliative toxin A, the causative agent of staphylococcal scalded skin syndrome (SSSS), has been developed. It is based on ammonium sulfate precipitation of the culture supernatant and hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. This procedure results in 87-fold purification of this toxin, which appears as a single band in sodium dodecyl sulfate-polyacrylamide gels.     

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 micro-Kjeldahl method for total nitrogen analysis

Summary A fast and simple micro-method for total nitrogen analysis in complex fermentation media is presented. Samples containing up to 50 ug nitrogen are digested in tubes with sodium selenite (in sulphuric-phosphoric mixture) plus hydrogen peroxide during one hour at 380°C. Ammonium liberated is measured without extraction by Berthelot colorimetric reaction.     

A simple and rapid method for isolating myelin basic protein

The conduction of impulses along axons of nerves is facilitated by the myelin sheath, composed of proteins and lipid. Myelin basic proteins (MBPs) are extrinsic membrane proteins that play an important role in the structural organization of the myelin sheath. In the central nervous system, MBPs account for 30-40% of total protein. The traditional method of MBP isolation involves the use of chloroform-ethanol, which would destroy the native form of MBP. A modified method for maintaining its native form was developed. The white matter of porcine brain was directly extracted by buffers containing different concentrations of sodium chloride owing to MBP solubilized at high concentration of NaCl. The MBP was further purified by cation exchange chromatography and buffers containing glycine and salts. Purified MBP were consistently obtained by this method.     

A rapid and simple method for plasmid copy number comparison

Summary A rapid, simple, and sensitive method for plasmid copy number comparison was developed. The extracted plasmids from the same amount of cells were subjected to agarose gel electrophoresis and the gels photographed. The photographs were processed by a Macintosh image analyser to enumerate the densities of plasmid bands. As a size reference, λ-DNA digested with a restriction enzyme was used. The densities divided by size of plasmids (base pair) would represent relative values of their copy numbers.     

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.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A simple and rapid solid-phase RNA sequencing method

A simple and rapid solid-phase RNA sequencing method has been developed based on Peattie's direct chemical method. 3'-Terminally labeled RNA was immobilized on DEAE-cellulose sheets and followed by specific modification with dimethyl sulfate, diethylpyrocarbonate, hydroxylamine (at pH 10 for the uridine and pH 5.5 for the cytidine reaction), and cleavage reaction with aniline. RNA fragments were washed from the DEAE-cellulose sheets using salt solutions, precipitated with ethanol, and separated by 15% polyacrylamide gel electrophoresis. Due to the complete removal of the impurities normally present in the solution method, the higher resolution of the sequencing bands and lower background on the autoradiograph make this solid-phase technique more efficient. This solid-phase technique is much faster and more convenient than the original method.