DNA extraction trouble
Posted 19 April 2009 - 10:45 PM
Posted 19 April 2009 - 11:22 PM
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.
Posted 21 April 2009 - 12:36 AM
Posted 21 April 2009 - 01:23 AM
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.....
Posted 21 April 2009 - 03:13 AM
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.
Posted 21 April 2009 - 03:31 PM
Posted 22 April 2009 - 07:12 AM
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.
Posted 22 April 2009 - 11:34 AM
Maybe there's a filtration technique you could use, with a filter large enough to let bacteria through, but not plant debris?