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.     

A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Extraction of nucleic acids from agarose gels

A rapid, simple and versatile method is described for the extraction from agarose gels of small plasmid molecules and DNA fragments generated by restriction endonucleases. The method may be used also for the extraction of RNA from agarose-urea gels. It is based on the partitioning of nucleic acid molecules into 1-butanol as their quaternary ammonium salts, leaving the neutral agarose in the aqueous phase. The nucleic acid is then recovered as the sodium salt by partition back into an aqueous phase. Nucleic acid samples were found to be unaffected by the treatment, as judged by their ability to be ligated, transformed, nick-translated, and used in an in vitro protein-synthesizing system.     

Densitometric determination of nucleic acids in gels after electrophoresis

A technique for the quantitative determining of nucleic acids after electrophoresis by double-wave densitometry in UV spectrum by scanning agar gel along both axes was developed. Optical characteristics of agar and acrylamide-agar gels are given. The range of quantities measured is 6 divided by 30 micrograms of DNA per gel; the standard deviation adjusted to 1 microgram is less than +/- 3%.     

An ethidium-acrylamide affinity medium for recovery of nucleic acids from free solution and from polyacrylamide and agarose gels

The simple preparation of an ethidium-bromide-based nucleic acid affinity medium is described. The medium is composed of an acrylamide matrix to which ethidium bromide is attached. Its use in preparative purification and fractionation of nucleic acids in solution and in electrophoretic elution of nucleic acids from gels is reported. Nucleic acids can be eluted from this medium with a buffered salt solution and concentrated by ethanol precipitation without persistent contamination with undesirable impurities.     

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.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.

A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.     

Visualization of proelastase in polyacrylamide gels

Our attempt to identify as many pancreatic enzymes as possible after their electrophoretic separation on polyacrylamide gels led us to search for a specific and simple method to localize proelastase.