An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters |
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Authors: | Floyd Taub E.Brad Thompson |
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Affiliation: | Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 USA |
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Abstract: | An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [3H]thymidine and [14C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter. |
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Keywords: | To whom reprint requests should be addressed. |
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