A method for the preparation of high molecular weight yeast DNA

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.     

Isolation of DNA from blood.

A rapid, economical method of DNA isolation from blood was developed that yields DNA suitable for Southern analysis and polymerase chain reactions without organic solvent extractions. Bovine DNA was prepared from peripheral leukocytes and nuclei using pronase E digestion and ethanol precipitation. This isolation method readily adapts to multiple samples. The DNA is characterized by high yield, solubility, lack of protein contamination, and ease of restriction endonuclease digestion.     

High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus

An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.     

Quantitative isolation of DNA restriction fragments from low-melting agarose by Elutip-d affinity chromatography

The use of a disposable affinity column and low-melting-temperature agarose for the quantitative preparation of DNA restriction fragments is presented. After electrophoretic separation of DNA, the band(s) are excised and the DNA/agarose melted in a low-salt buffer. After cooling, the DNA is bound to an Elutip-d affinity column. Fragments are eluted at high salt and concentrated by ethanol precipitation. Recoveries greater than 80% are achieved with purity suitable for most applications in molecular biology.     

Molecular cloning of PCR fragments with cohesive ends

Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids. Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized. The primers are chosen to span a known region of interest, and extended at their 5′-ends to include the desired restriction sites. Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers. A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s). Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize. Ligation products are used to transform competent bacteria. Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.     

Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs

A method is described for the isolation and purification of covalently closed circular (ccc) DNA from yeast (Saccharomyces cerevisiae). Spheroplasts are lysed at pH 12.45 which denatures linear but not ccc DNA. Next, the lysate is taken through a gentle high-salt-phenol extraction to remove single-stranded DNA. The ccc DNA, recovered by ethanol precipitation, can be further studied by agarose gel electrophoresis, can be cut with restriction endonucleases and can be used to transform Escherichia coli. This method efficiently purifies large (approx. 190 kb) and small (approx. 1.5 kb, TRP1-RI Circle) circular DNAs and thus has general applicability for isolation and purification of plasmids from yeast.     

Isolation of high molecular weight eukaryotic DNA with the use of potassium acetate

The simple technique for isolation of high-molecular eucaryotic DNA has been proposed. It includes cell or nuclei lysates by sodium dodecylsulfate in the presence of pronase, proteins precipitation by potassium acetate, DNA precipitation by ethanol. The DNA isolated by this technique is easily cleaved by restriction endonucleases and can be used for analysis of the unique genes by blot-hybridization. The yield of DNA is similar or somewhat higher than that in case of using the standard methods including phenol extraction or phenol-chloroform extraction.     

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.     

A quick and inexpensive method for removing polysaccharides from plant genomic DNA.

A quick and inexpensive method has been demonstrated to remove polysaccharide contamination from plant DNA. Isolated plant genomic DNA with polysaccharide contaminants was dissolved in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) with NaCl ranging from 0.5-3.0 M, then precipitated with two volumes of ethanol. Most of the polysaccharides were removed effectively in a single high-salt precipitation at 1.0-2.5 M NaCl. At 3.0 M NaCl, the salt precipitated out of solution. Purified DNA was easily digested by either HindIII or EcoRI and was satisfactory as a template for PCR. The results show that high-salt precipitation effectively removed polysaccharides and their inhibitory effects on restriction enzyme and Taq polymerase activity.     

Circular dichroism studies of the B goes to A conformational transition in seven small DNA restriction fragments containing the Escherichia coli lactose control region.

The B goes to A conformational transition caused by high ethanol concentrations was studied for seven DNA restriction fragments with overlapping and known sequences. Since the DNAs are homogeneous and range in GC content from 44-63%, they permit an evaluation of the influence of DNA sequence and base composition on the B goes to A transition. Moreover, their small size (80-301 bp) minimizes precipitation artifacts. The B- form spectra (in low salt) and the transition toward the C- form (in ethanol concentrations below the B goes to A transition) agree with prior measurements on chromosomal DNAs and are similar for all seven DNAs. At higher ethanol concentrations (80%), all fragments undergo a transition to the A- form as judged by the large increase of the positive CD band at 270 nm. Difference spectra among the fragments reveal minor differences between the A- form spectra. The ethanol concentration necessary to cause this transition is 72 +/- 2% for all fragments, thus excluding a preference of the CAP-, E. coli RNA polymerase-, or lac repressor-binding sequences for the A- form. The kinetics of the B goes to A transition in 80% ethanol are biphasic; the initial rapid transition is an intramolecular B goes to A form shift and the slower transition is an aggregation (but not precipitation) of the DNA     

Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes.

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.     

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.     

Mapping the B-A conformational transition along plasmid DNA

A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.     

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.     

A rapid method for preparation of bacterial plasmids

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.     

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.     

对ZnCl2沉淀DNA的方法的优化

通过优化影响ＺｎＣｌ２沉淀ＤＮＡ的若干条件,比较ＺｎＣｌ２和乙醇分别对大分子ＤＮＡ、寡核苷酸的沉淀效果,证实ＺｎＣｌ２法是一种具有很多优点的ＤＮＡ沉淀方法．     

A simple method for extraction of fungal genomic DNA

We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 microg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min(-1) in the presence of CsCl. The modified method yielded 1.5-2 microg of high molecular weight DNA per colony.     

The use of cetyltrimethylammonium bromide for the rapid isolation from Staphylococcus aureus of relaxable and non-relaxable plasmid DNA suitable for in vitro manipulation

Plasmid DNA was isolated from Staphylococcus aureus by a rapid method that depends on the precipitation of DNA from cleared lysates by cetyltrimethylammonium bromide at low salt concentrations. The method was validated by its ability to provide DNA for restriction analysis of a highly relaxable plasmid species that is not isolated by more traditional techniques. The DNA can be digested with restriction endonucleases and used for transformation without further purification. The method also provides the basis for analysing staphylococcal plasmids that display a high frequency of deletion after transfer. Simple modifications of the technique allow plasmid DNA to be isolated from other bacteria and the rapid purification of DNA samples before in vitro manipulation.