Separation of Choanoflagellate and Bacterial Genomic DNA
- 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94704, USA
- 2Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94704, USA
- 3Canadian Institute for Advanced Research, Toronto, Ontario M5G 1Z8, Canada
- 4Department of Biology, University of York, York YO10 5YW, United Kingdom
- 5School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
- ↵6Corresponding author (b.s.c.leadbeater{at}bham.ac.uk)
INTRODUCTION
Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. However, the preparation of genomic DNA from cultures of Monosiga brevicollis or other choanoflagellates can be complicated by the presence of abundant bacterial DNA. Indeed, bacterial DNA can represent as much as 90% of the total yield from a standard genomic DNA preparation. This protocol describes the use of a CsCl gradient to separate choanoflagellate genomic DNA from the DNA of its bacterial prey. This strategy works only when the G+C content of the choanoflagellate genomic DNA is significantly different from that of its bacterial food. The final DNA sample from this protocol is expected to be 90%-95% enriched for choanoflagellate genomic DNA and to provide an adequate template for genome sequencing projects.
