Date of Award:

5-1987

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Committee Chair(s)

Joseph K. K. Li

Committee

Joseph K. K. Li

Committee

Reed P. Warren

Committee

Jeffery K. Kondo

Committee

John R. Simmons

Committee

Dennis Welker

Abstract

Methods currently in use for the separation and identification of specific segments of nucleic acids involve long transfer periods or elaborate apparatuses and result in the production of a single blot. Contamination by organisms or enzymes is always a factor to be dealt with. An improved method for transferring nucleic acids from acrylamide or agarose gels for use in hybridization has been developed. This method uses NaOH as the blotting medium to improve the rate and efficiency of transfer to nylon membranes. As many as six blots can be obtained within one hour using this method. This method is effective for both viral double-strand RNA and single-strand RNA. By using 0.2 N NaOH as the transfer medium, and using nylon membranes for blotting, the nucleic acid appears to be covalently fixed to the membrane. These blots can be stripped of the probe and reused. In our studies with viral dsRNA, as little as 3.2 ng of total nucleic acid can be detected on the blot. This method provides a great improvement over previous methods for blotting and hybridization of both ss-and dsRNA and shows promise for use with dsDNA.

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