| abstract |
Methods and reagents for isolating polynucleotide molecules from uncultured microbial cells in an environmental sample, the method comprising: obtaining uncultured microbial cells from the sample, dispersing the cells from each other and from other components in the sample, purifying the dispersed cells via discontinuous gradient centrifugation wherein the cells are collected in an interface of the gradient, embedding the cells in agarose gel, to produce agarose gel blocks containing the cells, and lysing the cells within the agarose gel blocks, thereby releasing high molecular weight polynucleotide molecules from the cells. Also disclosed are methods and compositions for cloning the polynucleotide molecules so isolated, further comprising incubating the gel blocks with at least a suitable restriction endonuclease to partially digest the polynucleotide molecules, separating the digested polynucleotide molecules by pulse field electrophoresis, to recover fragments of polynucleotide molecules with size of at least 50 kb, ligating recovered fragments of polynucleotide molecules to a suitable cloning vector, and transforming a suitable host cell with the cloning vector containing the fragments of the high molecular weight polynucleotide molecules. |