The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns |
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Authors: | Barbara Koller Hajo Delius Hans Bünemann Werner Müller |
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Affiliation: | 1. European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, F.R.G.;1. Biologie III der Universität Bielefeld, Universitätsstrasse, 4800 Beilefeld F.R.G. |
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Abstract: | Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect. |
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Keywords: | Electron microscopy heteroduplex analysis effect of ultraviolet light phage T7 DNA restriction endonucleases |
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