Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

A protocol for DNA fragment extraction from polyacrylamide gels

A simple and efficient method of purifying linear plasmid DNA from contaminating DNA fragments is described. Both vector and insert containing plasmids may be used without extensive purification, in particular without cesium chloride centrifugation. Careful deproteinization with phenol-chloroform allows efficient restriction enzyme digestion. Fragment separation can be performed immediately after restriction endonuclease digestion in a single 6% polyacrylamide gel. Extraction of DNA fragments from the gel is easy and gives a good yield. The DNA may be used for ligation and transformation without further purification.     

A method for the recovery of nucleic acids from polyacrylamide gels

A method is described for the recovery of RNA from ethylene diacrylate cross-linked polyacrylamide gels. The RNA is recovered undergraded in good yields under mild conditions and is free of soluble polyacrylamide gels.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

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.     

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.     

Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels

A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.     

Visualising DNA: footprinting and 1-2D gels

The study of molecular recognition of DNA by natural and synthetic ligands has made enormous progress due in large part to the discovery and development of methods for separating DNA fragments by gel electrophoresis in one and two dimensions, and for characterizing DNA-ligand complexes by footprinting techniques.     

Rapid and inexpensive recovery method of DNA fragments from agarose and polyacrylamide gels by a cotton-wool column tube.

We developed a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA fragments ranging in size from 193-23,130 bp can be easily recovered within 2 hours by centrifugation through cotton wool from gel slices. The recovery rate of this method is 35% to 50%, when estimated for isolation of lambda DNA-HindIII fragments. We have also recovered 700-bp polymerase chain reaction (PCR) products using cotton wool tubes from electrophoresis on both a 0.8% agarose gel and a 6% polyacrylamide gel, in which satisfactory yields of more than 50% were obtained. The DNA thus recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.     

Strand separation of DNA fragments and their isolation from nondenaturing polyacrylamide gels

A technique for rapidly and quantitatively denaturing double-stranded DNA employing urea and moderate heat is described. The single DNA strands are resolved on high-percentage nondenaturing polyacrylamide gels from which they can be recovered for Maxam-Gilbert sequence analysis.     

Semi-dry electroblotting of DNA and RNA from agarose and polyacrylamide gels.

Semi-dry electroblotting of nucleic acids was originally of interest for its speed and convenience, but it had significant limitations in reliability of transfer. This work describes optimalization of the buffer systems and general conditions for use that enable the semi-dry method of nucleic acids transfer to yield quantitative, fast and reliable results.     

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.     

Improved estimation of DNA fragment lengths from Agarose gels

A simple, sensitive assay for prolylcarboxypeptidase (PCP) is described. It utilizes a radiolabeled substrate, benzyloxycarbonyl-l-prolyl-l-[³H]alanine, and the details of its synthesis are also reported here. The hydrolysis of the dipeptide substrate is linear with respect to time or protein concentration until 10% of the substrate has been cleaved. Kinetic analysis yielded a K_m of 4.7 mm. The assay can be used to measure PCP activity in small amounts of biological fluid, homogenized tissue or cultured cells. Measurements of PCP activity in various cultured human cells showed endothelial cells from umbilical veins to have the highest activity (1625 ± 151 nmol/mg/h) followed by endothelial cells from umbilical artery (1017 ± 46 nmol/mg/h), human foreskin fibroblasts (719 ± 39 nmol/mg/h), and pulmonary artery endothelial cells (352 nmol/mg/h).     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.