We are in the process of extracting bacterial DNA from water samples using QIAMP DNA mini kit. However, we could not see any visible bands on agarose gel after 16s hypervariable region amplification.
Can anyone suggest a protocol for sample preparation prior extraction? How can we get rid of inorganic matter dissolved in water with equipment/ methods available in a general laboratory?
Thanks
I think you should concentrate the sample by centrifuge that river water first. You could centrifuge the sample at 4000 rpm many times. After that you could extract the genome DNA using kit or by PCI method.
As I see, QIAMP DNA mini kit is used to extract DNA from tissues, swabs, CSF, blood, body fluids, or washed cells from urine. It wasn't designed to work with environmental sample where you have to deal with humic acid, organic and inorganic compound and many other unknown elements.
I used to extract total genomic DNA from water and soils sample using Mobio PowerSoil DNA extraction kit. Although this kit is used for soil sample, you can filter water sample and extract microbial DNA from the filtered paper. They also have water extraction kit but it is very expensive.
This kit is very good for environmental sample because it aids to remove many organic, inorganic, humic acid, cell debris and protein which can interfere your downstream procedure.
This link can direct you to the kit's information
http://www.cuny.edu/research/sr/undergrad-research/for-faculty/AREM/PowerSoil_DNA_Isolation_Kit_Instruction_Manual.pdf
Good luck
Thanh Nguyen
Hi
I think you should before all culture your water samples on fortified conventional bacterial mediums, provided that the culture process does not affect your analysis. This way helps you to avoid interfering materials and also increase the bacterial mass before DNA extraction.
Culturing the bacteria (dependent analysis) before performing DNA extraction is a good idea but you can't obtain all the information that you can possibly get. Because after all, the bacteria that grow on the media is all you can get. And with the laboratory condition (temperature and nutrient provided), a lot of time the cultivated bacteria are the unpopular ones and unable to represent the actual microbial population in the sample (actual environment where the sample obtained and bacteria grow is a lot of time harsher and more difficult to mimic).
@Sujani: Is it possible that you can share with us the purpose of you extracting DNA or the direction or your plan? It will be easier for us to help you.
Thanks everyone for the suggestions.
@Thanh- our plan is to do a culture independent, metagenomic analysis of water. Can't use the QIAMP DNA mini kit with some pre-treatment to the water sample? Will centrifugation or concentration of the sample work? Can you suggest protocols that can remove humic acids in the water in a laboratory setup?
hmm, i think if you want to remove humic acids in the sample, you can use CsCl2 density gradient centrifugation by making a series of concentration, and an increasing concentration toward the base of the tube. By doing high speed centrifugation (it need ultracentrifuge), the DNA will migrate toward the point where the density gradient of CsCl2 is the same with the DNA.
well i think that all DNA extraction kit use the same buffer and reagent generally, so it should be allright to use QIAMP DNA mini kit, except that this kit is specialized to extract DNA from eukaryote
take as big as you can sample volume and concentrate it with centrifugation tech. ex 50 ml sample can be spin for 20 to 30 min at 5000 to 8000 rpm.
then pellet should be washed with sterile double distilled water
washing step should be repeated two to three times so all deposited salts and other impurities will be cleared. now you can proceed for DNA extraction with Qiagen kit.
Hi Sujani Ariyadasa
I think the problem you have has to do with sample concentration. You nee to redefine your sample concentration where you can obtain more cell concentration then the first batch. Start changing this parameter first and after you can start thinking to go to a different direction. Remember to modify one parameter at the time to analyze the results. I think your kit of DNA extraction should be okay.
It is better to have thick bands then no bands at all.
Aloha Luis.
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