一个实用的制备EMBL4 DNA 的方法

以往人们通常用氯化铯梯度超速离心法、甘油梯度超速度离心法等方法纯化噬菌体。采用这些方法,虽然可以获得纯净的λ噬菌体颗粒。但需要昂贵的试剂和仪器。操作也冗长繁琐。我们采用并改进了Reddy的方法,首先用DE_(52)纤维素柱层析纯化λ噬菌体颗粒,然后用酚抽提,从提纯的噬菌体中分离DNA,这个方法简单快速,不需要氯化铯梯度超速离心,也不使用SDS、蛋白酶和核酸酶。     

四种土壤微生物总DNA的纯化方法的比较

比较了4种从土壤中直接抽提的微生物总DNA的纯化方法,实验结果表明1 %的琼脂糖凝胶电泳纯化方法及葡聚糖凝胶G 2 0 0离心层析纯化方法均不能完全纯化从土壤中抽提的微生物总DNA。若将直接抽提的总DNA先经葡聚糖凝胶G 2 0 0离心层析纯化,再用1 %的琼脂糖凝胶电泳纯化,则能取得较好的纯化效果。含2 %PVP的1 %琼脂糖凝胶电泳纯化,用DNA凝胶回收试剂盒回收后没有得到纯化后的土壤微生物总DNA。     

鸡蛋胚下表层卵黄DNA的提取方法

利用Ficoll-400不连续密度梯度离心将受精和未受精鸡蛋的胚下表层卵黄进行纯化, 显微镜观察表明卵黄球形态良好, 没有胚细胞的存在.然后利用较高浓度的蛋白酶K消化,较长时间的酚抽提,最后提取了DNA. 电泳显示DNA条带清晰.该方法简便快速,从每个鸡蛋的胚下表层卵黄可回收10 ng DNA.     

噬菌体T4诱导的脱氧核糖核酸(DNA)连接酶的分离和纯化

本文介绍一个从噬菌体T4诱导的大肠杆菌中纯化DNA连接酶的简法。这是从同一起始原料(噬菌体T4感染的大肠杆菌菌体)同时提纯三种酶(多核苷酸激酶,DNA连接酶及RNA连接酶)的步骤的一部分。这个方法包括以下几步：超声破碎菌体,抽提粗酶,用硫酸链霉素沉淀去除多核苷酸激酶和DNA．甩I)EAE纤素素(DE-52)柱层折将DNA建接酶与RNA连接酶分离开来,用磷酸纤维素(P-II)分步冼脱DNA连接酶,对在Tris-HCI,pH7.6中含50％甘油的缓冲液透析加以浓缩。最终得到高浓度(5500单位／毫升)和高纯度酶制品。     

一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用G8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl₂和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9.22 μg·g^-1,A260/A280为1.65,可适用于 PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0.67 ng·μl^-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.     

超滤过结合密度梯度离心纯化鲍曼不动杆菌噬菌体方法的建立

目的 探讨获得低浓度内毒素和高滴度鲍曼不动杆菌噬菌体的方法,为制备安全的噬菌体生物制剂提供参考.方法 用可截留100 kD以上分子量的超滤离心管浓缩噬菌体裂解液并滤出分子量约为10 kD的内毒素,然后用蔗糖密度梯度离心纯化噬菌体浓缩液;分别测定超滤前、超滤后和纯化后的噬菌体滴度,采用鲎试验测定超滤前后内毒素的浓度,通过SDS-PAGE分析超滤前后和纯化后噬菌体蛋白的纯度.结果 经超滤离心法噬菌体滴度从3.9×1010 PFU/mL提高至1.68×1012PFU/mL,并可去除99.2％的内毒素;超滤过结合密度梯度离心后的SDS-PAGE可清晰呈现7种蛋白,分子量为29～100 kD.结论 超滤过结合密度梯度离心是一种简便、快速浓缩和纯化噬菌体的方法,并可有效地去除裂解液中的内毒素.     

地衣芽孢杆菌的温和性噬菌体——Ⅱ.DNA分子生物学特性的研究

增殖Blp7、Blp11、Blp16这一类群噬菌体并抽提其DNA,用电镜法测定噬菌体DNA长度为62.75±1.47 kb,并证实它们与λ噬菌体相似,含有一个能使DNA环化的粘性末端。DNA经碱部分变性后,电镜观察可以识别Blp 7 DNA分子的R端与L端。用限制性内切酶EcoR1、BamHI酶切Blp7及Blp16 DNA,经琼脂糖凝胶电泳后,观察到DNA分子的多态性。     

碘化钾梯度离心在制备噬菌体DNA中的应用

通过氯化艳梯度离心除去细菌DNA及细 菌残渣来分离纯化噬菌体颗粒是一种方便而有 效的方法〔ZJ。其唯一缺点是氯化艳价格昂贵。 Blin等人〔13报道说,可用碘化钾梯度离心法从 琼脂糖电泳凝胶中回收DNA, Smith 13,发展了 Blin法,在碘化钾溶液中加人I MM Na2S20,以 防止碘离子的氧化,使DNA稳定不受干扰。 最近,我们把该法应用于分离纯化兄噬菌体的 重组体DNA,结果表明在噬菌体颗粒的梯度 离心纯化过程中,完全可以用碘化钾替代氯化 艳。     

检测噬菌体DNA法鉴别细菌的溶原性

根据前噬菌体的可诱导性,将细菌培养物经丝裂霉素C诱导,诱导液滤过除菌,经核酸酶处理和聚乙二醇(PEG 6000)浓缩,再用苯酚进行抽提。通过检测抽提物中有无DNA,以确定菌株的溶原性。实验证明从溶原菌诱导液中可提取DNA,同时表明该DNA确为溶原菌诱导出的噬菌体DNA,而非溶原性菌以同样方法不能取得DNAo用此方法,可以作为鉴别细菌溶原性的一个手段。     

DNA连接酶、RNA连接酶、多核苷酸激酶和DNA聚合酶的同时分离纯化

本文报道从T4amN82噬菌体诱导的大肠杆菌E.coliB 同时分离和纯化四种酶的一般方法。先用硫酸链霉素沉淀把RNA连接酶,DNA连接酶与多核苷酸激酶和DNA聚合酶加以分离。然后用DEAE纤维素柱层析把DNA连接酶与RNA连接酶加以分离,用DEAE-Sephadex-A50柱层析把多核苷酸激酶与DNA聚合酶加以分离。本文着重介绍T4DNA聚合酶的分离纯化。     

Isolation of lambda phage DNA by hydroxylapatite chromatography

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.     

PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping

A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling. This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of sequences cloned into lambda phage.     

Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts

A rapid and efficient method to purify lambda gt11 DNA is described. This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment. The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography. The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification. Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.     

An efficient method for preparing high quality DNA from plant DNA libraries in lambda phage

An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in vectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 g DNA was produced from 5 ml lysate obtained from agar plates.     

A high-throughput, low-cost method for the preparation of "sequencing-ready" phage DNA template

We have developed a simple and efficient protocol for the isolation of good-quality recombinant phage DNA useful for all downstream processing, including automated sequencing. The overnight-grown phage particles were effectively precipitated (without any contaminating Escherichia coli DNA and other culture media components) by adjusting the pH of the culture medium to 5.2 with sodium acetate, followed by addition of ethanol to 25%. The phage DNA was selectively precipitated with ethanol in the presence of guanidinium thiocyanate under alkaline pH, resulting in uniform quality and quantity of phage DNA. The quality of the phage DNA preparation was demonstrated by DNA sequencing that provided an average read length of >700 bases (PHRED20 quality). This protocol for plating, picking, growing, and subsequent DNA purification of individual phage clones can be completely automated using any standard robotic platform. This protocol does not require any commercial kits and can be completed within 2 h.     

Improved microfluorometric DNA determination in biological material using 33258 Hoechst.

A simple and rapid microfluorometric method is described for the determination of DNA in submicrogram quantities using 33258 Hoechst fluorochrome. A high degree of reproducibility was obtained using calf thymus and phage DNA, mouse liver chromatin, and HeLa cells homogenate preparations. None or very little interference by the routinely used preparation reagents or by the cellular components was found. Compared to other commonly used procedures this innovative and versatile technique can be conveniently applied to DNA microdetermination for the high sensibility/reproducibility ratio and can also be used without the need of previous purification steps.     

Purification of bacteriophage DNA by gel filtration chromatography

Two fast and effective methods for high-scale purification of linear phage lambda DNA and circular double-stranded M13 replicative form are presented. A substantial reduction of time is attained by avoiding the long-term CsCl gradient centrifugations and dialysis common to standard procedures. Biologically active DNA preparations, free of chromosomal DNA and RNA, are obtained by including a simple gel filtration chromatography as the last step of purification. Yields are comparable to those from previously described methods.     

Real-time PCR provides improved detection and titer determination of bacteriophage

The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

Typing of bacteriophages by randomly amplified polymorphic DNA (RAPD)-PCR to assess genetic diversity

The recent boom in phage therapy and phage biocontrol requires the design of suitable cocktails of genetically different bacteriophages. The current methods for typing phages need significant quantities of purified DNA, may require a priori genetic information and are cost and time consuming. We have evaluated the randomly amplified polymorphic DNA (RAPD)-PCR technique to produce unique and reproducible band patterns from 26 different bacteriophages infecting Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus lactis, Escherichia coli, Streptococcus thermophilus, Bacillus subtilis and Lactobacillus casei bacterial strains. Initially, purified DNA and phage suspensions of seven selected phages were used as a template. The conditions that were found to be optimal 8 μM of 10-mer primers, 3 μM magnesium oxalacetate and 5% dimethyl sulfoxide. The RAPD genomic fingerprints using a phage titer suspension higher than 10(9) PFU mL(-1) were highly reproducible. Clustering by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm correlated largely with genetically different phages infecting the same bacterial species, although closely related phages with a similar DNA restriction pattern were indistinguishable. The results support the use of RAPD-PCR for quick typing of phage isolates and preliminary assessment of their genetic diversity bypassing tedious DNA purification protocols and previous knowledge of their sequence.