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目的:为了达到批量提取质粒DNA的目的,在多次实验的基础上,建立一种经济、高效的质粒提取方法。方法:以pUC18、pET28b、pCAMBIA1304等3种质粒为材料,分别采用silica法和碱裂解法提取质粒DNA,通过质粒DNA浓度的紫外分光光度法定量测定、电泳分析和HindⅢ酶切鉴定,对两种质粒提取方法的效果进行了比较与评价;对silica法进行了改进和优化,进行大批量重组子的提取和验证。结果:silica法和碱裂解法提取质粒DNA效果相当,都可进行后续实验,但silica法具有经济、高效、无毒的优势。结论:silica法是一种简单、经济、高效的质粒提取方法,可用于批量质粒DNA提取。 相似文献
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DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method’s utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research. 相似文献
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Abhay K. Singh S. Chauhan G. S. Singhal 《Journal of plant biochemistry and biotechnology.》1995,4(1):51-53
We report here a simple and rapid method for the purification of chloroplast DNA (ctDNA) from wheat (Triticum aestivum). It utilizes an aqueous procedure, which does not involve at any stage running of gradients. Due to use of DEPC which inactivates DNases activated by EDTA, the DNase action on crude chloroplast preparation containing ctDNA is avoided. 相似文献
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HEINZ HAHN 《Physiologia plantarum》1975,34(3):204-207
A method is described for the isolation, purification and quantitation of free cytokinin bases and ribosides using ethyl acetate at pH 7.7 for the extraction. The extraction is almost complete (97.7%) as determined by using N6-(Δ2-isopentenyl)adenine-8-14C. The subsequent fast purification by chromatography on a standardized silica gel column in chloroform-methanol (7:3 v/v) is followed by thin layer chromatography (silica gel 60 F254) in chloroform-acetic acid (8:2 v/v). The recovery of N6-(Δ2-isopentenyl)adenine-8-14C after this two step purification was 78–82%. The efficiency of the method was determined by applying this procedure to N6-(Δ2-isopentenyl)adenine and N6(Δ2-isopentenyl)adenosine. Using gas liquid chromatography the recovery for N6-(Δ2-isopentenyl)adenosine was determined to be 61% and compared to 43% for N6-(Δ2-isopentenyl)adenosine, showing the suitability of the described method for gas liquid chromatography. 相似文献
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Ajay Kumar Hema Dawar Sudhakar Mathur 《Nucleosides, nucleotides & nucleic acids》2013,32(9):1575-1578
Abstract A simple, rapid and novel method for purification of the oligonucleotide using silica gel matrix is described. 相似文献
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The stability of palm oil was tested by subjecting it to elevated temperatures for different durations of time, viz; at 80°C for 150 hr and 60°C for 400 hr.The following results were obtained.(1) The absorption spectrum resembled that of carotenoid and this changed progressively with a rise in peroxide and carbonyl values during the first 80 hr at 80°C.(2) Peroxide values of Sabah palm oil were higher compared to Sumatra oil, there were marked increases in peroxide and carbonyl values of alkali refined oil as compared to crude oil. On the contrary, the residual color of crude Sumatra oil decreased considerably. Moreover, the steam emulsion number of alkali refined Sumatra oil was double the initial value after 400 hr. 相似文献
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Tiziana Bellini Mario Rippa Maurizio Matteuzzi Franco Dallocchio 《Journal of neurochemistry》1986,46(5):1644-1646
A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery. 相似文献
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涂楚国 《中国微生态学杂志》1990,2(4):57-61
本文报道快速简易、结果准确、应用广泛、效益明显,总大肠菌群和粪大肠菌群在15~18小时内同时检出,费用比部颁三步发酵法(GB)节省75~84%的大肠菌群检测新方法。笔者研究出 Lactose-Tryptone-Sodium Dodecyl sulfate-Trace Elements[简称 LTSE]培养基,加上几种鉴定验证试验的 LTSE 法,用来快速检测水质、食品、冷饮、餐具等各类样品703件,结果与国标法(GB)对照总符合率为99.3%;同时与美国《水和废水标准检验法》(16版1985年)新发展的现行标准检测粪大肠菌群的正式 A-1法相比较特异性强,灵敏度高,能提高检出率,并节约经费38.1%. 相似文献
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目的:针对分子育种工作的繁琐性及葱属植物次生代谢物较多的特点,研究一种利用普通试剂及简单仪器高效快速提取葱属植物DNA的方法。方法:对碱处理法稍加改进,在碱性环境中高温裂解细胞,将释放的DNA保存在Tris缓冲液中,用PCR检测提取质量并分析DNA的保存时间。结果:与用试剂盒提取的DNA相比,扩增产物无显著差异,利用此方法提取DNA并分析了13个洋葱品种的细胞质雄性不育类型;保存时间分析显示,DNA在常温下只能保存5d,在4℃或-20℃可至少保存2个月。结论:该方法方便快速、成本低、适于田间操作,得到的DNA可满足分子生物学实验的基本要求,可用于以PCR为基础的分子标记辅助育种。 相似文献
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M. A. A. Namboodiri M. J. Brownstein Pierre Voisin Joan L. Weller David C. Klein 《Journal of neurochemistry》1987,48(2):580-585
A two-step chromatographic procedure has been developed for the purification of ovine pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87), based on the principles of disulfide exchange and anion exchange. The enzyme from 20 ovine pineal glands can be purified about 500-fold in a day; recovery is about 5%. Polyacrylamide gel electrophoretic analysis of the final preparation shows four major bands; one appears to be arylalkylamine N-acetyltransferase. 相似文献
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A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4. 相似文献
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A rapid DNA minipreparation method was developed for rice and other plant species. This method uses an Eppendorf tube and 1-ml pipette tip to grind plant tissues, and requires only one transfer for DNA isolation. In a single day, one person can complete DNA isolation from more than 120 leaf samples. The yields of the DNA samples ranged from 2.3 to 5.2 g from 25–50 mg fresh leaf tissue. DNA samples extracted using this method from rice were completely digested with five restriction enzymes (EcoR I, EcoR V, Hind III, Mse I and Pst I) and were successfully used for AFLP and other PCR applications. 相似文献
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本文介绍一种简单快速分离质粒DNA方法。此方法有两个主要步骤。用这种方法分离的质粒DNA纯度高、无RNA,并可用于酶切、连接等操作。 相似文献
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The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B. By this procedure it is possible to obtain a highly purified enzyme preparation in a relatively short time with a 37.5% yield. 相似文献
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P. Nath A. P. Sane V. Bijola P. K. Trivedi J. Arora P. V. Sane 《Journal of plant biochemistry and biotechnology.》1993,2(2):117-120
We report here a simple procedure for the purification of the organelle DNA. Mitochondrial DNA from Sorghum and the chloroplast DNA from Populus and spinach were purified using this protocol. The method utilizes a quick centrifugation of the isolated organelle DNA through a two step CsCl density gradient for removal of small molecular weight nucleic acids which pose a major problem for getting clean restriction patterns. This method of purification can be adopted with any isolation procedure for organelle DNA. 相似文献