Preparation of bacteriophage lambda DNA using the TL-100 ultracentrifuge

A procedure for the preparation of DNA from bacteriophage lambda is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is of good purity and is easily cuttable by restriction enzymes.     

A rapid method for the isolation of circular DNA using an aqueous two-phase partition system.

A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction

A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction. The protocol is based on the purification of the amplified double-stranded product by polyethylene glycol precipitation, annealing of primer with template by a "snap-cooling" procedure and sequencing by the dideoxy chain termination method with the use of Klenow fragment or Taq polymerase. The limit of the size of PCR products that can be sequenced is also discussed.     

A new method of plasmid DNA preparation by sucrose-mediated detergent lysis from Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive)

A simple and cheap method of plasmid DNA preparation from both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) organism is presented here. In this method, in place of the high-priced chemicals lysostaphin and lysozyme which are commonly used for removal of cell-wall during plasmid DNA preparation from gram-positive and gram-negative bacteria, respectively, only sucrose has been used. Firstly, bacteria is treated with Trizma (pH 8.0) containing 100% sucrose (hypertonic solution). Due to this osmotic shock, protoplasm covered by the plasma membrane of bacteria possibly shrinks and becomes detached from the cell-wall. Osmotically sensitive cells thus formed, from gram-positive (S. aureus) and gram-negative (E. coli) bacteria, are finally lysed by the lysis mixture, containing brij 58 and sodium deoxycholate. The lysate is centrifuged at 15,000 rpm for 30 min to pellet the cell debris. The supernatant containing plasmid DNA is treated with either polyethylene glycol or isopropanol. The precipitate which contains plasmid DNA is dissolved in a buffer containing Tris, EDTA, NaCl, and sodium dodecyl sulfate (pH 8.0); thus protein is denatured and removed. Finally, RNA is removed by RNase treatment. The average yield of staphylococcal plasmid DNA as well as plasmid pBR322 from E. coli HB101 in 100% sucrose-treated preparations is greater than that of lysostaphin- and lysozyme-treated preparations. This method is applicable for both large-scale and small-scale preparations. The substrate activity for restriction enzyme, cloning, transforming ability, and electron microscopic profile of the plasmid DNA prepared by this method remains unaltered.     

A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.     

Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis.

A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.     

Integration of a perfusion reactor and continuous precipitation in an entirely membrane‐based process for antibody capture

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl₂ precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.     

Rapid purification of covalently closed circular DNAs of bacterial plasmids and animal tumor viruses

A rapid and simple purification of covalently closed circular (supercoiled) DNA from both bacterial clones (plasmids) and African green monkey cells (SV40) is presented. The method involves immediate treatment of lysed cells with sodium hydroxide, followed by neutralization and phenol extraction in high salt. After the extraction mixture is centrifuged, supercoiled DNA is found in the aqueous phase, the noncovalently closed DNA molecules form a white precipitate at the interphase, and proteins pellet. Contaminating RNA is eliminated from the aqueous phase by RNAse treatment and precipitation of the supercoiled DNA with polyethylene glycol. Residual polyethylene glycol is removed from the resuspended DNA by chloroform extraction. The purified supercoiled DNA is compatible with restriction enzymes, and is efficient at transforming both _χ1776 and HB101 bacterial hosts. Centrifugation in ethidium bromide-cesium chloride or sucrose gradients is not necessary. The method is virtually independent of the molecular size and gives good yields of supercoiled DNA. The technique is applicable to large-scale preparations and as a rapid “screening” procedure in which 20 to 30 samples can be easily purified within 5 to 6 h.     

Polymer-mediated compaction and internal dynamics of isolated Escherichia coli nucleoids

Nucleoids of Escherichia coli were isolated by osmotic shock under conditions of low salt in the absence of added polyamines or Mg(2+). As determined by fluorescence microscopy, the isolated nucleoids in 0.2 M NaCl are expanded structures with an estimated volume of about 27 microm(3) according to a procedure based on a 50% threshold for the fluorescence intensity. The nucleoid volume is measured as a function of the concentration of added polyethylene glycol. The collapse is a continuous process, so that a coil-globule transition is not witnessed. The Helmholtz free energy of the nucleoids is determined via the depletion interaction between the DNA helix and the polyethylene glycol chains. The resulting compaction relation is discussed in terms of the current theory of branched DNA supercoils and it is concluded that the in vitro nucleoid is crosslinked in a physical sense. Despite the congested and crosslinked state of the nucleoid, the relaxation rate of its superhelical segments, as monitored by dynamic light scattering, turns out to be purely diffusional. At small scales, the nucleoid behaves as a fluid.     

An extended boiling method for small-scale preparation of plasmid DNA tailored to long-range automated sequencing

Automated fluorescence sequencing depends on high-quality plasmid DNA, which is conveniently prepared by minipreparation procedures. While those procedures are effective for high-copy number plasmids, purity and yields of low-copy number plasmids are often not sufficient to achieve reasonable sequencing results. Here, we describe a reproducible and cheap procedure for the small-scale preparation of plasmid DNA, which is based on the original Holmes and Quigley protocol, comprising a boiling and two selective precipitation steps. Besides various other modifications, this procedure utilizes polyethylene glycol (PEG) precipitation as a key step to further purify plasmid DNA tailored to automated fluorescence sequencing. Independent of the plasmid size and copy number, the modified procedure yields plasmid DNA, which gives average reading lengths of 800 and more bases with a standard fluorescence cycle sequencing protocol. To demonstrate the efficiency and reproducibility of the method, sequencing data of various human interleukin-6 gene variants cloned in different vectors are presented. This procedure offers an economical alternative to commercial miniprep kits, utilizing silica resins or anion-exchanger matrices and, moreover, is more reliable and consistent with respect to reading lengths and accuracy in automated fluorescence sequencing.     

Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given.     

Isolation of human complement component C3 from small volumes of plasma

A method for the isolation of 8-10 mg of human C3 from 20 ml of plasma is described. The procedure is simple and rapid with excellent yields in hemolytic activity (74%) and antigenic activity (68.7%). It consists of polyethylene glycol precipitation, DEAE-Sephacel chromatography, and immunoadsorption. The final product is free of contaminating proteins as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The simplicity and speed of this procedure allow for the continual availability of hemolytically active C3.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.     

Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase

Recombinant Escherichia coli (pATBX 1.8) secreting extracellular xylanase was used as a model system to study the application of an aqueous two phase system for extractive cultivation. An increase in the polymer concentrations from 6 to 20% in the polyethylene glycol phosphate aqueous two phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of xylanase in the top phase. However, varying phosphate concentrations from 8 to 16% decreased both the phase volume ratio and the partition coefficient of xylanase. The polyethylene glycol (6%) and phosphate (12%) system was found to be optimum for extracellular cultivation of E. coli, where extracellular xylanase was selectively partitioned to the top phase giving a purification ratio of above 1.0. The process was extended to a semicontinuous operating mode at the optimal condition, wherein the top phase containing xylanase was recovered and the surviving cells were recycled together with the new top phase. The maximum recovery of xylanase was obtained after 12 h in the top phase with a twofold increase in the specific activity as compared to the one obtained in the reference fermentation. In the present work, we report for the first time the use of the two phase system for the extractive cultivation of recombinant E. coli (pATBX 1.8) with the purpose of obtaining a simple and inexpensive separation procedure and achieving the maximal extraction of xylanase to one phase.     

Cooperative transitions of isolated Escherichia coli nucleoids: implications for the nucleoid as a cellular phase

The genomic DNA of Escherichia coli occurs in compact bodies known as nucleoids. Organization and structure of nucleoids are poorly understood. Compact, characteristically shaped, nucleoids isolated by the polylysine-spermidine procedure were visualized by DNA fluorescence microscopy. Treatment with urea or trypsin converted compact nucleoids to partially expanded forms. The transition in urea solutions was accompanied by release of most DNA-associated proteins; the transition point between compact and partially expanded forms was not changed by the loss of the proteins nor was it changed in nucleoids isolated from cells after exposure to chloramphenicol or from cells in which Dps, Fis, or H-NS and StpA had been deleted. Partially expanded forms became dispersed upon RNase exposure, indicating a role of RNA in maintaining the partial expansion. Partially expanded forms that had been stripped of most DNA-associated proteins were recompacted by polyethylene glycol 8,000, a macromolecular crowding agent, in a cooperative transition. DNA-associated proteins are suggested to have relatively little effect on the phase-like behavior of the cellular nucleoid. Changes in the urea transition indicate that a previously described procedure for compaction of polylysine-spermidine nucleoids may have an artifactual basis, and raise questions about reports of repetitive local structures involving the DNA of lysed cells.     

A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones

An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.