What is the best way to isolate genomic DNA from water? Using the ready kit centrifuge a sample of water to obtain the sediment of microorganisms or maybe filter water and isolate DNA from the sediment accumulated on the filters?
this is an interesting question!!!!
i think it is better to filter the water from visible pollution. then spread the water on the prokaryotic medium culture. so, you can recognize microorganisms and also extract genomic DNA with high concentration to conform several molecular analysis
the best
It depends on what type of water and what type of organisms you are interested in. Filters and centrifuges will be less effective for viruses and phages. Salt water has different organisms than fresh water. Clean cold water has fewer organisms than swamps or muddy waters, etc.
https://www.ncbi.nlm.nih.gov/pubmed?linkname=pubmed_pubmed&from_uid=18845550
Thank you for your reply. I have a sample of fresh water from the river. I am interested in bacteria and fungi. But not individual species, but whole microbiome.
this is an interesting question!!!!
i think it is better to filter the water from visible pollution. then spread the water on the prokaryotic medium culture. so, you can recognize microorganisms and also extract genomic DNA with high concentration to conform several molecular analysis
the best
Most of the protocols that I am familiar with are for seawater samples, however, from my understanding it is the same for fresh water. A 0.22 micron filter media 47 mm diameter is OK (polycarbonate) and filter with a vacuum pump. Try to keep the pressure low and this will increase the volume that you can filter. The filter media is then cut into strips and added to a DNA extraction kit. This is the most important point, add the filter media to the extraction kit the same way you would add soil to the extraction kit (I have used Qiagen PowerSoil) some may use Qiagen PowerBiofilm or others. That is something that you will need to validate yourself. As for volume the more the better, however, usually 0.5 to 2.0 litres will work. this should be somewhere between half to one hour. Once again a trial will be necessary as it depends on the sample! Also, have a think about the level of sanitation that you would like. As always, check relevant research papers for their methodology.
Good luck!
The first step needed is to filter the water sample from all kind of undissolved impurities. Then you can isolate the prokaryotes using specific media and then go for characterization and DNA isolation of the colonies. DNA isolation of bacteria using ROSE (Rapid One Step Extraction) Method will be most suitable, easy and efficient one. For fungal DNA isolation you can follow the CTAB method or other specific methods that you can get from following references:
1. Plant Pathology & Quarantine 5(2): 74–81 (2015)
2. Nucleic Acids Research, Volume 20, Issue 9, 11 May 1992, Pages 2380, Doi: ttps://doi.org/10.1093/nar/20.9.2380
3. Lett Appl Microbiol. 2000 Jan;30(1):53-6 (Using SDS)
Start by culturing in enriched media or one that is selective to the organisms of interest
The better procetures are filteration, cultivation and isolation of organisms, then extration of DNA.
It depends which organisms are you targeting, you can use the DNA by PowerSoil® DNA isolation kit (Mo Bio, Solano Beach, CA), then PCR (Aoyagi et al. (2015)) and then metagenomic analysis, you will be able to identify all the microorganims present in the water. If you will culture bacteria from the water and then extract the DNA, it is fine too but just remember there are some bacteria which are unculturable.
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