Rapid transfer of DNA from agarose gels to nylon membranes.

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.     

Rapid isolation of high-molecular-weight DNA from agarose gels

We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.     

Purification of DNA from agarose gels

Summary We describe a rapid and easily reproducible modification of the freeze-squeeze method of separating DNA from agarose gels. Our method involves slicing out the agarose gel portion which contains the DNA of interest, freezing this gel slice at –20°C, then centrifuging the frozen slice in a filtration unit which contains a cellulose acetate filter. The agarose is retained on the filter and the filtrate contains the DNA. DNA purified in this manner could be completely digested with restriction endonucleases and completely ligated with DNA ligase, without further purification. The percentages of recovery for various sizes of linear and plasmid double-stranded DNA ranged from 57 to 69%. The procedure takes less than 30 minutes to perform.     

Simple DNA elution from agarose gels

We developed a simple DNA elution method from agarose gels. After electrophoresis of DNA in an agarose gel, the DNA fragment to be recorved was excised out of gel with a scalpel. The excised gel was placed in the middle of small Parafilm piece, and the Parafilm was folded over the gel piece. Using the petriplate, or thumb, the gel piece was pressed between the Parafilm. Upon squeezing, the DNA inside of the gel gets extruded along with the buffer. The droplets were collected with a pipet. The DNA was then purified by conventional phenol: chloroform extraction method. Typical yields are greater than 50% as determined by UV absorbance.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Recovery of DNA segments from agarose gels

After electrophoresis, DNA can be efficiently recovered by solubilization of agarose gels with NaClO₄, followed by retention of DNA on glass fiber filters. After removal of the NaClO₄ by ethanol, the DNA can be extracted with a low salt buffer.     

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.     

A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels.

Defined RNA fractions can be recovered from low gelling temperature agarose gels by a combination of agarose melting at 65°C and phenol extraction. By this approach RNA molecules up to a size of 37 kb can be eluted undegraded with a recovery of 60–90%. The method is applicable also to DNA and the eluted DNA can be correctly cleaved with restriction endonucleases as shown for λDNA using EcoRI.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.     

Rapid electroelution of nucleic acids from agarose and acrylamide gels

The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [³H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., ¹⁴C-labeled DNA) relative to the elution of “reference” DNA (i.e., ³H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ～0.190 and ～0.017 min^?1.     

A method for eluting DNA in a wide range of molecular weights from agarose gels

We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs obtained by our method recommend it for routine laboratory use.     

A rapid method for extracting DNA from agarose gels

A method for obtaining high recovery of deoxyribonucleic acid (DNA) from agarose gels using an agarase extraction procedure is presented. This DNA is physically intact and biologically active. The DNA obtained with this procedure should be useful for a wide range of applications.     

A low molecular weight alkaline proteinase fromConidiobolus coronatus

Summary An extracellular, low molecular weight alkaline proteinase (alkaline proteinase B) has been purified to homogeneity from the culture filtrate ofConidiobolus coronatus (NCIM 1238). A 12-fold purification was achieved with a specific activity of 29,760 u/mg. The enzyme had an optimum pH and temperature of 9.7 and 45°C respectively. It was most active towards casein and had a molecular weight of 6,800, the lowest reported so far. It was stable between pH 6.5–7.5. Alkaline proteinase B is a serine proteinase. It showed an esterolytic activity on N-benzoyl-L-tyrosine ethyl ester (BTEE) and was successfully used to resolve the racemic mixture of D, L-phenylalanine and D,L-phenylglycine and can thus potentially replace subtilisin Carlsberg in resolving the racemic mixture of amino acids.     

Electroelution of DNA and protein from polyacrylamide and agarose gels

An electroelution method is described for the recovery of DNA and protein from agarose or polyacrylamide gels. The samples to be electroeluted are compartmentalized in a modified microcentrifuge tube fitted with dialysis membranes. This procedure is simple, rapid, inexpensive and efficient. Within 30 min to 2 hrs, the recovery of the sample is nearly quantitative. DNA fragments recovered can be directly subjected to DNA sequence analysis or enzymatic reactions after ethanol precipitation. Proteins can also be recovered after separation by acrylamide gel in the presence or absence of detergents and be ready for further analysis.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.