separate plant protoplast from bacteria‏ prior DNA extraction - (Oct/29/2012 )

Hi all

My work is focused on microbes-plant interaction using metagenomics. I have extracted DNA from Leaves and fruits and around 95% of the sequences i obtained were from the choroplast and mitochondria from the plant, and actually i am interested in the bacteria growing in this samples.

does someone know a protocol to separate the plant protoplast from the bacteria to use it before performing the DNA extraction?

Thank you so much for your help
Iratxe

by the way...congrats for the forum, it is really interesting!

You could try density gradient centrifugation, but as chloroplasts and mitochondria are approximately bacterial size, it may not be very efficient/easy.

I don't know if this is a feasible solution, but here are two papers describing dielectrophoretic separation of different cell types from a mix (yeasts, bacteria, and plant cells):

Markx GH, et al. Dielectrophoretic characterization and separation of micro-organisms. Microbiology (1994), 140, 585-59.
Markx GH and Piethig R. Dielectrophoretic separation of cells: Continuous separation. Biotechnology and Bioengineering (1995) 45:4 337–343.

I don't know much about it so can any one tell me about DNA as well as RNA Extraction in detail.
_________
total rna