蓝藻质粒DNA提取方法的改进

以含pMD-489-TNF重组质粒的丝状体鱼腥藻7120和单细胞聚球藻7002为材料,比较了SDS-碱裂解法和SDS法在蓝藻质粒提取中的效果,并对SDS-碱裂解法应用于蓝藻质粒提取作了一些改进和实验条件的优化。     

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。     

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.     

一种快速、有效、经济的提取酵母质粒的方法

目的：建立一种经济有效、快速简便、稳定的提取酵母质粒的方法。方法：用葡糖苷酸酶消化酵母细胞壁以获取原生质体,然后采用碱裂解法裂解原生质体以获得质粒。结果：与采用商品化离心柱法试剂盒所提取的质粒相比,用该法获取的酵母质粒在PCR分析及转化效果方面没有差异。结论：建立了一种经济有效、快速简便、稳定的提取酵母质粒的方法。     

一种碱裂解菌液直接电泳快速筛选重组子的方法

目的：从碱裂解法提取质粒DNA的原理．经过实验摸索获得一种快速、经济、结果可靠的菌液直接碱裂解电泳筛选重组子的方法。方法：不需提取质粒DNA．只需将细菌培养液碱裂解后直接进行普通琼脂糖凝胶电泳分析,就可以快速筛选出转化重组子。结果：结果和提取质粒酶切鉴定鉴定结果一致。结论：经实验证明菌液直接碱裂解电泳筛选重组子是一种快速、经济、可靠的方法。     

直接用酚和氯仿快速提取质粒

质粒的提取是分子克隆技术中最关键的步骤之一,在对大量样品的抽提和筛选时工作量很大。提取质粒的方法很多,有碱裂解法、煮沸法等「１」,所用试剂较多,耗时较长,这些方法均要用酚和氯仿抽提以去除菌体蛋白,以免影响酶切分析。Ｂｉｊｉ,Ｔ,Ｋｕｒｉｅｎ等（１９９４）提出用乙醇溶菌法快速提取质粒,作者试验多次结果均不理想,经过改进直接用酚和氯仿溶解菌体来快速提取持粒,得到了满意的结果。用该法提取质粒ＤＮＡ,产量     

用于质粒DNA规模化生产的大肠杆菌发酵培养基的筛选

为降低质粒DNA的生产成本,在标准LB培养基的基础上,利用国产试剂配制成十种大肠杆菌液体培养基,以pEGFPC3、pcDNAlacZ和pcDNKLYZ质粒转化的JM109和DH5α大肠杆菌为指示菌进行小规模发酵培养,定时采样测量OD600值及质粒产量,获得一种高性价比培养基。用该培养基培养重组大肠肝菌,绘制生长曲线,并于其对数生长中期进行42℃诱导。结果表明经42℃诱导后,重组大肠肝菌JM109和DH5α的质粒产量均有提高,重组JM109的产量比重组DH5α约提高20％,为低成本、大规模生产重组质粒提供了良好的技术保障。     

侧孢芽孢杆菌质粒提取方法的探究

以能够降解有机磷农药的两株侧孢芽孢杆菌BL-21和BL-22为研究对象,分别采用碱裂解法、试剂盒提取法和SDS法对侧孢芽孢杆菌BL-21和BL-22的质粒进行提取,并通过凝胶电泳和紫外分光光度法对提取结果进行分析,试验结果证明,适合侧孢芽孢杆菌BL-21和BL-22的质粒提取方法是SDS裂解法,该方法提取的质粒大小为10kb,且该方法提取的结果稳定,质粒的产量和质量均符合分子生物学实验的要求。     

快速小量提取质粒DNA的两种方法

碱裂解法是进行分子克隆实验中质粒DNA制备最为常用的方法。在实际实验操作过程中,对该实验方法进行了改进,改进的碱裂解法解除了所提质粒DNA中RNA污染及RNase的可能影响,快速提取法使阳性克隆的筛选更加迅速,快捷。两种方法的改进使实验更加高效、简单、实用。     

一种简便快速筛选重组子的方法

目的：根据碱裂解法抽提质粒DNA的原理,研究应用快检缓冲液方法挑取重组子克隆,从而获得简单易行,快速方便,节省时间,提高工作效率的筛选重组子的方法。方法：不需抽提质粒,只要将菌落接入快检缓冲液后直接进行普通琼脂凝胶电泳分析,就可以快速筛选出重组子。结果：结果和提取质粒酶切鉴定结果一致。结论：经实验证明用快检缓冲液方法筛选重组子是一种简单易行,快速方便,节省时间,提高工作效率并且可靠的方法。     

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.     

Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.     

Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A

Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH ( approximately 12-12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18DeltalacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.     

Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.     

Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications.

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.     

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.     

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。     

Purification of plasmid DNA by tangential flow filtration

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.     

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.     

一种改良的质粒DNA小量提取法

对碱裂质粒小量法进行了改进,并且在提取过程中增加了ＬｉＣｌ处理。实验证明这种方法结果稳定,提取的质粒ＤＮＡ产量高、质量好,符合大多数分子生物学常规实验的要求。