simultaneos DNA purification - (Sep/26/2007 )
Hello, I am a rookie molecular biologist with a problem between hands that is the following:
I need to perform simultaneos purification of gDNA from bacteria, yeasts and fungus in the same plate to be later sequenced and identified.
I thought of resuspending from the plate in any buffer like PBS, adding SDS and boiling in order to lyse cells and running the extract in agarose gel to purify the genome bands, but I'm not sure if that's the best method. If anyone could lend me a hand I would be grateful.
well SDS and boiling is quite a hard treatment for genomic DNA purification. You may find kits designed for tha, and they probably give better yield and quanlity too.
Yeah, I suppose it's a bit hard for gDNA, but since I have those three kind of microorganisms with so different cell wall structures I have to find an efficient method for all of them.
The two main problems I have are:
1.- How to lyse all those cells types
2.- Once lysed, if genomes could be separated in and agarose gel to be further purified and sent to sequencing
Thanks for your help!!
oh well i though you had these organisms separated...
well sorting these 3 genomes?... yast has 13 chromosomes and their size is different...
Fungus i don't know.
I know that yeasts can be broken with french press.
I presume you don't want to subculture the three different types (keeping them pure) and then prepare the gDNA using the most efficient technique for each type of organism. It would make the later identification much simpler.
Yes, I've considered that option, that would be the very best, but since some species may be hazardous, we want to manipulate them as less as posible, so lysing the cells at their arrival would simplify the process very much