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DNA extraction trouble - (Apr/19/2009 )

I am interested in extracting microbial genomic DNA from disease-infected plant leaves. The problem is i get too much plant DNA contamination in the final DNA pool. Is there any method to seperate microbial (bacterial & fungal) genomic DNA from plant DNA? I dont know what the microbes are present in the diseased leaves.

-deeps-

Depends on what you want to do and what you want to analyse.

If you have no idea what you are looking for: I would suggest to make a clone library of your gene of interest, test all clones with (nested, insert specific) plant-primers, exclude positive ones and sequence the rest.

If you know what you are looking for: build specif primers for your organism. This might take some optimisation work.

-gebirgsziege-

Right now im following the same thing...cloning all fragments and testing for microbial inserts..and 60% are plant inserts. its time consuming and expensive. What about ultra centrifugation? will it seperate microbial DNA from plant DNA? my colleague rejected this coz sheared plant DNA will probably have the same size as microbial DNA and will definitely come into the picture even after ultracentrifugation...is there no other technique to seperate plant DNA from microbial DNA other than PCR??

-deeps-

You can use restriction enzymes, which cut the plant DNA but not the microbial DNA in your region of interest.
Otherwise: use different primers....primers which fit your target better than the ones you are using currently.....its quite some testing to do but in the end it pays off.....

-gebirgsziege-

hmmm.... I feel that separation of microbe and plant genome should be done before the DNA extraction stage. Would it be possible to wash off the microbe from the plant tissue? Would culturing the microbe be possible?


hmmm... (again). it might be possible to use Pulse field gel electrophoresis to separate out microbial genome from plant nuclear chromosomes. (You need to lyse whole cells in gel plugs). The sample will still be contaminated with chloroplast and mitochondrial DNA.

-perneseblue-

Depending on the microbial pathogen, the GC content of the DNA may be dramatically different. This difference is widely used in the phytoplasma community as a basis for enriching bacterial DNA from their infected plant samples, since the gc content of the phytoplasma genome is only 25% or so.

-phage434-

BIG IF here,

if u're looking for what microbes are on the leaves, u can extract DNA and then use Universal 16s primer specific for eubacteria perhaps to amplify out the 16s rRNA. then for sure whatever come out mmust be of bacterial origin.

-hanming86-

is this an intercellular plant pathogen, or one found on the plant itself (outside)?

Maybe there's a filtration technique you could use, with a filter large enough to let bacteria through, but not plant debris?

-HomeBrew-