DNA isolation from all bacterias in drinking water - (Aug/27/2006 )
I want to isolate DNA from the bacterias (both Gram positive and Gram negative bacterias) in drinking water. Do you have any protocols which is good for this purpose? THank you very much!
I would enrich first let say niutrient broth. There are established protocols to isolate specific bacteria from water.
As far as i concern, depending on the source of the water, you can find thousands of different microorganisms, so, i dont know if the isolation is used in this case unless you want to do PCR for the identificationof them
Filter different amounts of water (probably a lot for drinking water) on 0.22 µm filters and then put the filters on selective medium for selection of some particular species. If you want to analyse the community DNA use the filter for DNA extraction and then whatever community analysis technique you want (DGGE...)
if you go for enrichment and then dna extraction, then you are leaving out the unculturable
organisms.so, the best bet would be to go for the metagenome of the orgamisms present in your sample. the protocol involves the use of a strong extraction buffer containing ctab.
hope this helps.
I would use something more physical like beat beating. This is best for lysis of gram negatives and positives. Gram positives tend to be more resistant to chemical lysis. Of course, bead beating does shear DNA a bit so it would not be good for fosmid libraries but perfect for PCR. So if you wanted to make a 16S library it would be fine. Do a search in the literature for bead beating DNA extraction methods.