A simple method for extraction of fungal genomic DNA

We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 microg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min(-1) in the presence of CsCl. The modified method yielded 1.5-2 microg of high molecular weight DNA per colony.     

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

Methodology: simplified preparation of a DNA ladder using PCR

Serving as a DNA molecular weight standard, the DNA ladder has been widely used in molecular biology applications. We developed a simple method for the preparation of a DNA marker, which involves designing primers to amplify 100- to 1000-bp DNA fragments using lambda DNA as a template for polymerase chain reaction, followed by extraction with phenol/chloroform, precipitation with ethanol and mixing. Fragments of 100- to 1000-bp DNA were successfully amplified; the sequences showed 100% identity with lambda DNA. This prepared DNA marker displayed clear bands, indicating that it can be used for molecular studies.     

介绍一种简单高效的植物总RNA提取方法

在液氮中研磨小麦幼叶和不同发育时期的种子,经含0.1% SDS和0.1%十二烷基肌氨酸钠(LDS)的尿素缓冲液裂解后,醋酸钠和氯仿沉淀变性蛋白质,异丙醇沉淀核酸,溶解后经2.5mol/L LiCl沉淀总RNA,洗涤后就可得到高质量的总RNA,其OD260/OD280 为2.05~2.10,28S和18S RNA带清晰,叶片总RNA还可得到23S和16S RNA带,产率可达5mg RNA/10g材料。当使用含1% SDS和1% LDS的尿素缓冲液裂解材料时,则可用于DNA的分离提取,其分子大小可达50~100kb以上。 Abstract:Wheat leaf and seeds at different development stages had been squashed in liquid nitrogen,then lysised by urea buffer which contains 0.1% SDS and 0.1% LDS,denatured protein had been removed by NaAc and chloroform precipitation,total RNA was further purified by LiCl.The RNA we obtained had sharp bands of 28S and 18S after agarose gel electrophoresis,23S and 16S RNA bands can also be seen clearly in leaf RNA extract,the value of OD260/OD280 of RNA was 205~210.5mg RNA can been isolated from 10g leaf of wheat.This method can also been used in high molecular weight DNA isolation but the concentration of SDS and LDS must be increased to 1%.     

小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA.

In mammalian cells DNA synthesis is more complicated than in prokaryotes and less well understood. Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human fibroblasts) with [32P]orthophosphate and found that, besides high molecular weight DNA, a species of low molecular weight DNA, approximately 450 bp in size, became efficiently labeled. The short DNA was labeled first, and in pulse-chase experiments the labeling was transient. The isolated small DNA fragments (RNase A-treated) were phosphorylated by T4 polynucleotide kinase specific for polynucleotides with 5'-OH ends. A polynucleotide kinase phosphorylating these DNA pieces was also detected in nuclear extracts of the cells. Treatment with alkaline phosphatase removed most of the 32P label incorporated into the small DNA in vivo. Labeling with deoxyribonucleosides did not reveal these fragments. We hypothesize that the low molecular weight DNA represents Okazaki fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki fragments. This would imply a novel step in DNA synthesis. We also show that depriving cells of polyamines reversibly blocks synthesis of high molecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.     

植原体DNA提取方法的改良

在总结多种植原体DNA提取方法的基础上 ,发展了一种提取植原体DNA新方法。用此方法提取的DNA经琼脂糖凝胶电泳检测到大于 15kb的DNA主带 ,基本无DNA碎带 ,不用RNase处理 ,也无RNA干扰 ,OD2 60 / 2 80 值显示产物纯度较高 ,无需任何处理 ,即可以作为模板扩增     

Species of small DNA molecules found in mitochondria from sugarbeet with normal and male sterile cytoplasms

Summary Mitochondrial DNA and RNA were isolated from a range of normal and cytoplasmically male sterile sugarbeet varieties and breeding lines. When these nucleic acids were analysed by electrophoresis on agarose gels it was found that mitochondria from normal sugarbeet contain DNA species of sizes 1.3 kilobase pairs (kbp), 1.4 kbp and one of two other species of sizes 1.45 kbp and 1.5 kbp, in addition to their DNA of relatively much higher molecular weight. In contrast mitochondria from cytoplasmically male sterile sugarbeet contained only one of these DNA species, that of 1.5 kbp. Treatment with DNaseI, RNase and nuclease S1 showed that these species consisted of supercoiled circular DNA. It is not known whether the lack of the two smaller DNA species causes cytoplasmic male sterility, or whether the two traits are associated by chance.In addition it was found that mitochondria from some individual sugarbeets contained one or more types of high molecular weight RNA molecules which were probably double stranded. Also mitochondria from some sugarbeet lines and varieties contained a series of DNA molecules with molecular weights in the range 2 to 10 kbp. Neither these DNA molecules nor the RNA molecules were apparently correlated with cytoplasmic male sterility.     

Rapid isolation of total RNA from mammalian tissues

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.     

Oligonucleotide mapping of non-radioactive virus and messenger RNAs.

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.     

鱼肝线粒体DNA的简易提纯方法

本文报道一种简易分离纯化线粒体DNA(mtDNA)的方法。即以差速离心或蔗糖梯度离心制得线粒体后,用RNase除去RNA,可得纯mtDNA,或用凝胶过滤、或低熔点琼脂糖法纯化之。所获纯mtDNA可用于限制性酶切图谱的组建和其片段的克隆。此法可避开冗长的昂贵的超速离心,故可为普通实验室所采用。文中还讨论了mtDNA纯化和得率的因素。     

A rapid and inexpensive method for preparing E. coli plasmid-DNA

A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure E. coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-Cl chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 micrograms for pBR-322 DNA, and 250-500 micrograms for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation does not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days.     

Acetone preservation: a practical technique for molecular analysis

In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.     

RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization

An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus.     

哺乳类(兔)红细胞分化因子的纯化及特性分析

本文报道从哺乳动物(兔)红细胞分离、提纯红细胞分化因子(ErythroidDifferentiation Factor,简称EDF及对其性质的分析。按Bishop方法给新西兰兔注射盐酸苯肼(2.5％)0.3mL/kg体重/日连续6days致贫血,收集血液用生理盐水洗去白细胞后,得到的网织红细胞用冻融法使细胞裂解,用乙醇、氯仿选择性变性除去血红蛋白,凝胶过滤,连续经DEAE-纤维素及CM-纤维素柱层析,取得较原裂解液纯化了31 000倍的活性物质EDF,产率为9.1％,分子量15kD,在SDS-PAGE电泳图中为单一蛋白带。EDF对热相对稳定,100℃处理10min仍保持一定活性,对蛋白酶敏咸而对DNA酶I及RNA酶不敏感。EDF对体外培养细胞的生长抑制作用已在体外实验体系得到验证。     

提取航天诱变向日葵种子总RNA的一种有效方法

目的:从航天诱变向日葵种子中提取高质量的总RNA.方法:采用改进的SDS法,提取缓冲液与氯仿同时作用液氮研磨材料后,用酸酚-氯仿抽提一次,经LiCl过夜沉淀、DNase I处理、1/2体积的无水乙醇沉淀多糖,最后加入1/10体积的醋酸钠和2倍体积的无水乙醇沉淀总RNA,用琼脂糖凝胶电泳与紫外分光光度法测定产量与纯度,用...     

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.     

Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.     

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.