Hi Thanh

The yeild might be less  if there is small noumber of microbe in the water samples. You may need to filter more volume of samples to get a substantial amoutn of DNA for further analysis.

bye

I'm thinking loud over here because its not my area of expertise, but will it be possible to provide a general growth medium and amplify the number of microbes in your water sample and then maybe extract the DNA?

Hi Gargi,

Many times such common growth is not feasible for all the microbes present in such a community. This is the basis of analysis such as metagenome sequence analysis. It may be the concern of Thanh...

Thank you for your responses, Ajay and Gargi.

I am trying to extract the total DNA with which I can use as template to run PCR and qPCR so I guess growing the bacteria at the first place doesn't work in this case (but I did that to get the control).

And my samples are running out. I only have 50 mL to start out and I've already used over half of them...

From of my view, water equal to blood as a liquid and they are harbored many microbes in very low levels, for example some microbes find in blood in low levels such as chronic infection. thus, recommend use of blood extraction kits which are available by several companies.

Or, according to the microbes you want to detect them, take water sample into specific culture media and then will extract DNA from colonies

HI Thanh,

I am not sure whethe you have tried the PCR based analysis or not using the extracted  DNA you already have. Since you are planning PCR and qPCR based approaches and as they are highly sensitive approaches...your planned analysis may work properly....just give it a try with few analysis. ...If you are targeting certain specific regions of the microbes you can further enhance the sensitivity of qPCR by designing specific fluorecently labelled primers/probes instead of SYBR green based assay...If at this moment there is nothing you can do with the samples..it is better to go ahead and see if PCR/qPCR based assays will work ....all the best

bye

Filtering water might cause the loss of floating eDNA, hence I would suggest you to use salt-precipitation method instead. But of course, it depends on what sort of organisms you are targeting. This paper can be useful for the technique I mentioned: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0041781

Personally, I use phenol-chloroform extraction, which I think gives more DNA yield than the commercial kits. It may be more time-consuming, but at the end you can obtain substantial amount of DNA. 

Best of luck!

@Mr. Saini (I'm sorry for calling you by your first name without your permission): Thank you for your suggestion. I tried to run the PCR, without a lot of DNA in the samples, I still can see faint bands at the right size (but with some bands at the neg control too which I have never had before). I reran it and will check for amplification today. I let you know how it goes. If it appears with faint bands (which I normally have anyway), with good negative control. I will run the qPCR next Monday.

@Mr. Burzynski: I wish that I could have more samples to trouble shooting and find the best way to do it but it was collected for a while and I only have ~ 30 mL left. I really want to find a way to extract the DNA with a better yield and not cost whole lot of sample.

@ Ms. Baloglu: Thank you for the paper, I will have a deeper look at it. I hope it helps. I think of phenol-chloroform extraction before but haven't tried it yet. How much of water sample do you normally need for this procedure?

Hi Thanh,

It is good you have started working on PCR and could go ahead with your analysis. If there is less DNA in your existing sample..there is nothing you can do about and also you don't gain anything by saving it. It is better try to do some PCR/qPCR analysis and get   some results out of it. It is still not clear what types of regions/genes you are targeting and what is the purpose of the experiments...is it just identification of microbes present in the sample or something more.....if the objective is first one then you may  choose high copy regions in the genome (such as rRNA gene regions) which may amplify even with less amount of DNA...and another important point would be ...which primers and what PCR conditions ar eyou using...will they work for the organisms you are using or some optimizations would be needed....faint bands in PCR may also result due to inefficient PCR amplification...so this is also impotant.

Just to clarify, AJAY is my first name and Saini is my family name...so you don't have to be sorry for that and there is not need for permission to us emy first name or family name..

best regards

bye

Hi

You can filter your water with suitable filter paper depending on your need.

Subject the filter paper for the DNA extraction

Regards

Hi Thanh Nguyen,

You can inoculate microbes by using nutrient agar plate. After 24 hours incubation colony will be formed and can be extracted DNA from them as per you requirement. Thanks. 

Hi all.

As you know to study of bacterial community in water, recovery of bacteria from filter member is critical step. Does any one know how can I get best result to recovery of entrapped bacteria in filter? Best,

Why are we going for filtering the water? Why not just centrifuge? We need all bacterial cells in the water so if we centrifuge we will get it. Am I right in saying that?

Rifat Z Ahmed

Centrifugation may cause suspension of some free floating bacteria while filteration will surely get all bacteria on paper, may be that is why filteration is more appropiate method.

Hello, Thanh Nguyen

You can extract DNA from water with DNeasy Powerwater Kit (Qiagen) and you use filter with 0.22µm.

Hello Thanh Nguyen:

I'm certain that at this point that it's all water under the bridge (so to speak), but Patrick Ferreira above has indicated the kit that you would use for best results. In addition, should you do further sampling, you should minimally get 500 mL of water and up to 5 L of water, depending on the source. For environmental water samples that contain very few microbes (such as water from caves), 5 L is common. For most other water samples, 1 -- 2 L seems to be the common amount needed. I filter in a laminar flow hood using sterile bottle filters (0.2 or 0.22 µm), then cut out and remove the filters, which can then be placed folded or cut up into the DNeasy Powerwater Kit (if you are only doing water samples) or the DNeasy Powersoil Kit if you have other types of samples to compare. The DNeasy Powerwater Kit is best, if you only have water samples, as (according to the techs at Qiagen), it will give you the largest yields.

Dear Ellen Lark

Do you know what is the best way to store the 0.22 um filters after filtration? I want to collect more water samples over time and then analyse them all together. Can I put the filter in a 15 or 50 mL falcon tube and store at -20 C. Or is it necessary to add anything?

EDIT: my colleague stores the filters in petri dishes (small ones can be sufficient as well) which is much more convenient than falcon tubes (although it will take up more space).

Best regards, Carina

I store all my unextracted samples in a -80C freezer (no matter what they are). After they are extracted and I have the community genomic DNA I will store them at -20C.

After filtering water samples, I usually store them at 4oC for s short period of time and -20oC for longer. There would be no occasions where you would need to use -80oC unless they are stored for years

Paper filters tend to have large pore size (Whatman 1 ~ 11um; see https://en.wikipedia.org/wiki/Filter_paper) and even worse they have depth, so extracting your DNA even if you did capture your bacteria is tricky.

What you could do is use 0.45 or 0.2 um filter, such an nylon (does not capture the cells. If you have cloging problems place a GF/C filter (glass fiber C; pore size ~1.2um) and extract both together.

Even better, although expensive, get Aluminum Oxide filters. after you filter you can grind them and treat them as soil.

Hi, I used DNeasy Powerwater Kit (Qiagen) for extracting DNA from 0.22µm filter. I addition to low concentration, I am also having problem with the quility of DNA.I got 15-25 ng/uL DNA and the value of A260/A230 is 0.205-1.0 which is supposed to be more than 2.0

Any suggestions?

hello, some of you have tried to extract DNA without using a comercial kit? like traditional protocols? Do you know where I could find it? Thanks

Cecilia Mlewski - have you tried CTAB extraction with Phenol Cholorform clean up?

Nicole Pietrasiak no!!! I will try it! thanks!!

I ultracentrifuged the sample at 130000g for 30mins followed by spindown at 14000g for 10mins to get a pellet. Then extracted DNA by qiagen DNA mini kit.