碱裂解法提取重组质粒DNA及PCR验证

目的:筛选获得高质量质粒,为进一步克隆、测序奠定基础.方法:采用常规的碱裂解法从大肠杆菌重组质粒中提取质粒DNA,核酸检测仪测定提取质粒DNA产量和纯度,并用前期DDRT-PCR时采用的引物进行PCR验证性实验,琼脂糖凝胶电泳检测,并对电泳图谱加以分析和鉴定.结果:利用常规的碱裂解法提取重组质粒,通过酚和氯仿的抽提,可以有效地去除蛋白质杂质,用含有Rnase抑制剂的无菌水溶解质粒提取效果最好,得到的质粒DNA无RNA污染,纯度比较高,OD260/OD280比值介于1.8～2.0之间,OD260/OD230比值大于2.0,经PCR反应验证后与预计相符.结论:通过该方法获得的重组质粒纯度和浓度都比较高,且PCR验证与预计相一致,可以满足后续分子生物学实验的要求.     

一种经济高效的用Silica提取纯化质粒DNA的方法

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。     

农杆菌质粒DNA提取方法的优化

目的：为了减化操作过程,减少药品对操作人员有直接或间接危害,提高农杆菌质粒DNA的产率和实验结果的稳定性。方法：对常规碱裂解法进行了改动,改进的方法通过加大菌液的收集量,利用LiCl沉淀RNA,简化操作流程和严格反映环境条件,并且在操作过程中去除了酚、氯仿等对人体有害的试剂。结果：提取时间缩短,结果稳定,产率在1．5μg／μL以下。结论：用这种方法提取的质粒可以满足大多数分子生物学常规实验如DNA的酶切、PCR鉴定的要求。     

一种大规模筛选单克隆及提取质粒的改进方法

利用α-互补(X-gal IPTG)从cDNA库中筛选特异克隆,以此去除含有未插入片段和插入片段小的克隆。运用展库的方法,通过辅助噬菌体对库中多个载体的切割,将载体以质粒的形式转化到大肠杆菌中;同时对质粒DNA碱裂解提取方法做了改进,建立了一种简单实用的大量提取质粒DNA的方法。该方法利用特定的蛋白质吸附膜吸附提取过程中的蛋白质,从而去除了使用酚-氯仿抽提蛋白质的复杂过程,减少了质粒DNA的损失,是一次性获得大规模质粒DNA的有效方法。获取的质粒DNA样品在纯度和浓度上都可以满足测序、PCR及Southern杂交等分子生物学实验的要求。     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。     

苏云金芽孢杆菌大型质粒DNA的小量提取

以苏云金芽孢杆菌库斯塔克亚种、猝倒亚种及以色列亚种为材料,介绍了低拷贝大型质粒DNA的小量提取方法,该法采用PEG 6000进行质粒纯化,省略了苯酚和氯仿抽提过程。实验证明,该法结果稳定,提取的质粒DNA产量和质量均符合大多数分子生物学实验的要求。     

重组大肠杆菌碱裂解方法的改进

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。     

用于哺乳动物细胞转染的高纯度质粒DNA的制备

目的：建立简便高效、成本低廉和安全无污染的高纯度质粒提取方法。方法：在乙酸铵方法的基础上加以改进,主要改进之处在于增加了用聚乙二醇纯化质粒的步骤,并对溶液Ⅱ和溶液Ⅲ的成分和具体实验参数也做了合理的改进,以最少的步骤,充分去除了残存杂质,保证了质粒的超纯状态。结果：用本方法提取的质粒与用QIAGEN plasmid midi Kit提取的质粒在理化指标上没有差别,对哺乳动物胞具有同样的转染效率。结论：本方法可完全取代QIAGEN公司的试剂盒用于提取超纯质粒。     

用酚-氯仿直接从克隆中提取质粒DNA

目的:对用酚-氯仿直接从克隆中提取质粒DNA的方法进行了研究。方法:采用不同次数的酚-氯仿抽提,在不同RNaseA作用温度及作用时间下提取了三种质粒。结果:用酚-氯仿抽提2次,RNase A45℃作用8min,质粒纯度(OD260/OD280)为1.85左右、产量大于0.8μg/克隆。结论:本法不但快速,且所提质粒均能满足后续实验如酶切鉴定、测序等方面的需要。对于大批量重组克隆的筛选尤为适合。     

从全血中提取基因组DNA方法的对比研究

目的:建立从全血中提取基因组DNA的方法,并评价提取DNA的产量和质量.方法:分别采用传统酚-氯仿抽提法和试剂盒法制备基因组DNA,比较这两种方法提取DNA的产量、纯度、稳定性以及方法本身的优缺点、费用等.结果:试剂盒法简单快速,但其制备的DNA产率、纯度、稳定性低于酚-氯仿抽提法.结论:采用酚-氯仿抽提法可以提取较高质量的基因组DNA,适用于下游的分子生物学实验.     

Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.     

一种简便易行核酸疫苗质粒纯化工艺的开发

介绍了一种成本低、步骤少、简单易行的质粒纯化制检工艺。该工艺选择优势产生超螺旋质粒的大肠杆菌菌株以无蛋白质培养基进行发酵罐培养,采用碱裂解法,对质粒制备过程中所用的层析吸附材料、核酸结合溶液、去除内毒素等杂质的方法和浓缩等步骤进行了实用性改进,并建立了相应的检定方法,所得质粒的纯度达到临床级要求。     

Improving the recovery of qPCR-grade DNA from sludge and sediment

DNA extraction is often considered as the limiting step of most molecular approaches in ecology and environmental microbiology. Ten existing DNA extraction protocols were compared for recovery of DNA from sludge and a modified version of the protocol described by Porteous et al. (Mol Ecol 6:787–791, 1997) was determined to be the best method for recovery of DNA suitable for PCR. In this respect, it appeared that the commonly used guanidine isothiocyanate could impair the quality of the extracted DNA unless its concentration is lowered. Second, conditioning the samples as liquors as opposed to pellets critically impacts the outcome of the extraction. The suitability of the modified Porteous protocol for quantitative PCR applications is demonstrated in a series of experiments showing the absence of interfering coextracted inhibitors and the linear correspondence between the concentrations of input target DNA and PCR product. Interestingly, it is also shown that the nature of the environmental matrices affects the recovery yield of both circular plasmids and chromosomal DNA, resulting in an apparent fluctuation of the plasmid copy number per cell. This means that quantitative data obtained by PCR remain comparable as long as they apply to an identical target sequence extracted from a similar environment and amplified under the same conditions.     

Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

Direct detection of nif-gene sequences of Enterobacter agglomerans in soil

Abstract: Specific nif sequences of Enterobacter agglomerans plasmid pEA9 were detected in total DNA recovered from soil 70 days after its inoculation with the bacteria, when these were no longer culturable on agar medium. For this, a modified method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was necessary.     

一种提取质粒DNA的改良方法

本文详细介绍了一种改良碱裂解法提取质粒ＤＮＡ的方法,该法采用ＮＨ４Ａｃ代替苯酚和氯份的抽提过程,得率高,质量好,完全达到了分子生物学常规实验的要求,如酶切、连接、转化大肠杆菌、ＰＣＲ等,甚至用于序列测定和植物遗传转化,该法重复性好,操作简单、实用．     

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.     

A modified mini-prep method for economical and rapid extraction of genomic DNA in plants

Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of 700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based markers.