Photographic quantitation of DNA in gel electrophoresis (original) (raw)
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The Kurume Medical Journal, 1991
To quantitate DNA from photographs, correlations between chromato-scanned (in the fluorescence mode) and virtual areas from white DNA bands on photographs were examined. A linear correlation between the two measurements with coefficients higher than 0.89 was observed. The dynamic range of the fluorescence detection yielded good linearity for a wide range of amounts of DNA (ƒÊ-ng) on the photograph.
Hoechst 33258 staining of DNA in agarose gel electrophoresis
Journal of Microbiological Methods, 1986
A staining procedure for agarose gel electrophoresis using Hoechst 33258 has been optimized. A concentration of 5 x 10-s M allows the immediate visualization of as little as 1 ng of DNA with a standard UV illumination system. The major advantage of this method is the elimination of the carcinogen ethidium bromide used in most DNA visualization methods for gel electrophoresis.
Analytical Biochemistry, 2006
Ethidium bromide (EtBr) 1 is a substance that has widely been used in biochemistry, molecular biology, and histochemistry for several decades . For example, it is applied for DNA or RNA staining in agarose and polyacrylamide gels as well as for staining of histological samples. The structure and properties of EtBr are well known , and the mechanism of its interaction with nucleic acids has been described previously . EtBr intercalates between DNA or RNA bases and binds up with them, allowing one to view in UV light the bands of these nucleic acids that are formed in agarose and polyacrylamide gels as a result of the electrophoresis process. The EtBr technique is characterized by the rapid action and simplicity that determine its broad application . In the current work, the possibility of protein staining in polyacrylamide gels by EtBr after preliminary gel soaking in trichloroacetic acid (TCA) solution is demonstrated. The method is as simple as that of DNA staining and by sensitivity is comparable to the standard Coomassie method applied to protein gels. EtBr can be used for immediate evaluation of results obtained by electrophoresis of mixed nucleic-protein samples. The suggested mechanism of EtBr interaction with denatured proteins is discussed.
Analytical Biochemistry, 1977
Ultraviolet sources with output wavelengths of 254,300, and 366 nm were compared for detection of ethidium bromide-DNA complexes in acrylamide gels. The 254-and 300-nm sources were both much more sensitive than the 366-nm source. The 254-nm source produced a great deal of photodamage, photonicking and photodimerization, and photobleaching, while the longer wavelength sources cause little damage or bleaching. The 300-nm source is clearly the most suitable source, providing high sensitivity and a relatively low amount of photodamage and photobleaching. The examination of DNA on polyacrylamide or agarose gels using fluorescent staining with ethidium bromide (EB) is convenient and effective (1). Ultraviolet sources for exciting the EB-DNA complex are commercially available, with output at either 254 or 366 nm. Each of these sources has its shortcomings. The 254-nm source produces photodamage in the nucleic acid and causes photobleaching of the complex. The 366nm source is relatively inefficient and gives a weak fluorescence of the complex. The excitation maximum of an EB-DNA complex occurs in the range of 300 nm, and fluorescent lamps that emit in this range are commercially available. We have constructed an ultraviolet source using these 300-nm lamps in conjunction with a filter that passes the ultraviolet and blocks visible wavelengths. This system should be optimal for the fluorescent detection of EB-DNA complexes. In this paper, we compare the 300-nm source with commercially available 254-and 366-nm sources as a means of detecting EB-stained DNA bands on an acrylamide gel. METHODS Construction of a 300-nm source. The 300-nm source was constructed using eight 8-W 300-nm fluorescent lamps (RPR-3000 A).l These lamps
Quantification of DNA samples by Ethidium Bromide Spot Technique
bioRxiv, 2018
Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. More importantly, we hope that our approach can help other labs optimize this protocol for their own experimental setup. Adoption of this technique may help enable development of iGEM teams in resource limited environments and laboratories which do not or cannot employ a satisfactory method for determining DNA conc...
DNA staining in agarose and polyacrylamide gels by methyl green
Biotechnic & Histochemistry, 2018
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.
Ethidium Bromide Spot Test Is a Simple Yet Highly Accurate Method in Determining DNA Concentration
2006
A commonly used method to measure the amount of DNA in nucleic acid preparations is to use UV irradiation. However, if the DNA sample contains impurities, measuring UV irradiation is misleading. A quick way to estimate the amount of DNA in such samples is to analyze spot densities created by UV-induced fluorescence emitted by ethidium bromide. The amount of DNA is proportional to the amount of fluorescence. In many references, the spot density analysis is recommended for rough estimation of DNA concentration and is done by visual examination and comparison of the densities of each spot with the densities of DNA standards. Herein, we used a computer-integrated software to test the reliability of such a method, and showed that the analysis is highly reliable and simple.
Electrophoresis, 1995
Commercial automated gel electrophoresis apparatus : Application to DNA, band dispersion, nonlinear Ferguson curves, and isolation Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/ DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 pg load for a fullscale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching. Electrophoretic band dispersion was not found to be due to diffusion since zone spreading in the absence of the electric field under otherwise identical conditions does not proceed measurably. For the discontinuous buffer system of Wiltfang et al. (Electrophoresis 1993, 12,352-366) the resolving power, in terms of theoretical plate equivalents, was approximately threefold higher compared to that in the Trisborate-EDTA (TBE) buffer under otherwise identical conditions. Two electrophoretic runs of the apparatus with different gel concentrations in its eight channels suffice to define nonlinear Ferguson curves, provided that the free mobility is measured by capillary electrophoresis. The commercial automated apparatus excels as a preparative device by allowing one to monitor the qualitative and quantitative success of electroelution.