Date of Award

Spring 2001

Document Type

Thesis

Department

Life & Environmental Sciences

First Advisor

John Addis

Second Advisor

Anthony Szpilka

Third Advisor

Gerald Shields

Abstract

With greater emphasis being placed on molecular approaches to systematics, it is becoming increasingly important to be able to extract DNA from alcohol-preserved specimens, such as those maintained in museum collections. As a preliminary step to DNA sequence analyses o f alcohol-preserved freshwater sponge specimens, this study compared the purity and yield of genomic DNA isolated from alcohol-preserved specimens by three different DNA extraction methods. One method (standard method) made use of a proteinase K digestion in the presence of detergent and EDTA, followed by extraction with phenol-chloroform. A second method (CTAB method) involved proteinase K digestion in the presence of cetyltrimethylammonium bromide (CTAB), followed by extraction in chloroform. A third method employed a cell lysis solution containing guanidium thiocyanate, followed by extraction in phenol-chloroform. Analysis of isolates indicated (1) no difference in purity or yield between DNA extracted from alcohol-preserved and frozen samples and (2) no difference in purity among methods, although yield was highest using the CTAB method. Preliminary evidence also suggested that all DNA preparations contained appreciable amounts o f RNA.

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