According to standard protocol, DNA in agarose inserts is prepared by first embedding the cell in agarose. This is then incubated in the required enzyme (lysozyme, lysostaphin, or zymolase) depending on the cell type (bacterial or plants), for spheroplast formation. Subsequent treatment of the spheroplast with proteinase K allows the isolation of large genomic DNA in agarose suitable for pulse field gel electrophoresis. An efficient and rapid method of preparation of spheroplast is described. In this method a low concentration of enzyme required for spheroplast formation was added before embedding the cell in agarose, which facilitated the digestion of cell wall by the enzyme and allowed use of a low amount of enzyme. Digestion of DNA in agarose inserts prepared by this method, with rare cutting restriction enzyme and pulse field gel electrophoresis, showed that the quality of DNA was as good as obtained by the standard method.