- Hits: 1652
The technique employed to extract and amplify the DNA from single microbial cells for the microbial dark matter study is now detailed in a Nature Protocols. We describe all necessary steps from sample preservation and preparation, to single cell separation, fluorescence-activated cell sorting (FACS), cell lysis and finally the whole genome amplification.
The procedure takes four days, can be perfomerd in a standard molecular biology lab, and yields up to a microgram of DNA per single microbial cell, an amount considered “suitable” for PCR amplification and shotgun sequencing. And please note, that although the protocol was applied to microbial cells, it can also be adapted for microbial eukaryotes and mammalian cells.
Rinke C., J. Lee, N. Nath, D. Goudeau, B. Thompson, N. Poulton, E. Dmitrieff, R. Malmstrom, R. Stepanauskas, and T. Woyke, “Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics,” Nat. Protocols, vol. 9, no. 5, pp. 1038–1048, May 2014.