Plasmids DNA from Slreptomyces sp. IQ45 were isolated and identified by gel electrophoresis using different isolation methods and separation techniques Optimization of protoplast formation was achieved when the bacterium was grown in S-medium or modified NYM in presence of 1.5% glycine for 48 hrs. Under such conditions, 99% of protoplast was formed. Heat and phenol treatments were found to have a critical role in plasmid extraction when rapid procedures were used for plasmid screening. The obtained results showed the superiority of such treatment in comparison with the methods using salt precipitation of chromosomal DNA and proteins. Results of conventional gel electrophoresis showed the presence of (7kb) cccDNA. Orthogonal field-alternation gel electrophoresis resulted in identification of giant linear plasmid of (240 kb). All eleclrophorcsis techniques were modified to increase the efficiency of DNA fractionation by agarose gels. These modifications included ,the separation of the buffer in the positive electrode(s) region from that in the negative electrode(s) region, the making of the buffer in direct contact with the edges of the gel, and the placing of the gel over a cooling box. Results obtained from these modifications were, shortening time required for electrophoresis, homogenous transfer of electric field through the gel, and increasing the force applied on the DNA molecules in the sample which made those DNA molecules travel more efficiently through the gel.