Don't worry. All u need to do is filtering 2.5-5L water on a 10cm diameter 0.22μm (pre-filtration using 10μm membrane is recommended) membrane then follow the manufacture instructions for Gram+ protocol.

I would strongly suggest you first concentrate your water sample using a tangential filtration system, using an appropriate cutoff filter to concentrate the microbes of your interest.

Once this is done, use a MoBio kit for soil, it should take care of the various inhibitors that can potentially inhibit your downstream application like next gen sequencing.

I would suggest against Qiagen kits for this specific purpose.

The Qiagen DNA Stool Mini Kit produces good results. It has a reagent (in tablet form -'Inhibitex') that binds inhibitors in a specific part of the protocol.

Hello there,

I read recently a very interesting paper on the impact of different commercial kits on a metagenomic study (it was gut microbiota, but still interesting for you I think).

Having done metagenomic studies on water, it is likely that you'll find many actinobacteria, that are usually tougher to break than the others. This is to be kept in mind to be sure to get the whole picture of the diversity.

Good luck!

Thanks all, I am still in the look out for the best DNA extraction method using commercial kit. There are so many in the market, making it very difficult to choose. So far I am getting good vibes from qiagen DNA stool kit, FastDNA.

Jay: I am looking through the MoBio powewater though.

Vincent: Yup that paper kind of enlightened my knowledge

Thanks Philip and Fang Liu

FastDNA is fast and results in good quantity, yet the quality is not very ideal as Qiagen. Mobio give you the clean DNA with low quantity according to my experience.

Hi Nytia. It's difficult to tell you the best option. There are a lot of options and they will work differently depending on the type of your sample. Some water samples could have a lot of organic matter and humic compounds and in this case it would be better to use a kit for soil. You won't need to do tangential filtration in most of the times because normally it has at least 100000 bateria/ml and a few liters will be enough.Besides the sample, you should think about the storage of the material prior to the extraction. Keypoints:

- water composition

- storage

sterivex is a nice think to use or a filter to hold the bacteria after remove the big particulates. For both options you can do homemade protocols or dedicated kits (e.g. Epicentre and Mobio)

Nelson

Thanks Nelson.

Between if I were to extract DNA from filter membrane by using Qiagen Stool mini kit, how do I do so? I have not come across any modified protocol to extract the bacteria cells from a filter paper. Anyone know any modified protocols to do that.

Dear Fang Liu, I tried checking for the gram positive modfied extraction but none was mentioned about filter paper.

can anyone help?

Thanks so much