ABSTRACT
Metagenomic deoxyribonucleic acid (mgDNA) library is constructed using Pushpam et al. method. Soil contains diverse microbial populations of both culturable and non-culturable bacteria, therefore, mgDNA was isolated by the indirect extraction method. A small-insert metagenomic library was constructed in the cloning and expression vector pET-32a. Functional screening of metagenomic libraries is a powerful approach to identify and assign function of novel genes and their encoded proteins without any prior sequence knowledge. The transformation of recombinant and non-recombinant plasmid is done by J. Sambrook and D. W. Russel method. Metagenomic DNA fragments of 0.5-3.0 kb size were ligated into the restricted pET-32a plasmid vector using T4 DNA ligase enzyme. DNA fragments were resolved in 0.7% (w/v) agarose gel electrophoresis. After the separation by Agarose gel electrophoresis, the gel is examined under UV transilluminator and the gel slice containing DNA fragments ranging about 0.5-3 kb is separated using sterile scalpel. The recovered DNA fragments are used for library construction.
