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How to determine the volume of lisis buffer and DNA extraction buffer when the a

DNA extraction bioflim

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#1 cerbatana

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Posted 12 November 2011 - 06:08 AM

Hi, I hope the title of this topic is not misleading.

I'm trying to extract DNA from the anode biofilm of a microbial fuel cell (MFC) for the first time. I'm writing a protocol based on papers and the book Molecular Cloning and I've managed to adapt current protocols to the equipment available in our lab. However, while some papers dealing with microbial DNA extraction specify at some extent the volume of every solution used for cell disruption and DNA purification from proteins and carbs, most (including those where anode biofilm DNA is extracted) don't, but I assume it's in function of the amount of biological material available for extraction (whether it is 100 g of soil or 0.3 g of a sample of biofilm). The anode we use for our MFC is rather small in comparison with the anodes used for microbiological characterization (44 square centimeters of superficial area; 0.22 g dry weight; carbon cloth) and I've proposed to scrape off half of the surface for the acquisition of a biofilm sample (I'm not planing to remove the entire biofilm since we need the MFC working just after the removal of the sample) so it's very likely that the amount of biofilm obtained is even smaller than those used by other researchers.

So the question I have is, how do I decide what volume of reagents to use? If some protocol uses for example 0.3 g of biofilm and I obtain 0.15 is it correct to assume that half of the volumes used by the other researchers will work for me? How did they find the correct volumes to work with in the first place? trial and error?

I have the correct concentrations of the solutions, but when it comes aobut the volume all I can do is guess.

Thank you in advance for your help.

Edited by cerbatana, 12 November 2011 - 06:38 AM.


#2 bob1

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Posted 13 November 2011 - 02:22 PM

These things are scalar to some extent, but I think you will find that even working with half the amount of biofilm and the full amounts of the solutions will also work fairly well. If you struggle to get DNA, which I doubt will be a problem, you can add a carrier such as yeast tRNA, salmon sperm DNA or linear polyacrylamide.

Basically you will just have to try it and see, that is usually how these things have come about. You might get a methods paper out of it, if you can work it out well.

#3 cerbatana

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Posted 13 November 2011 - 10:27 PM

These things are scalar to some extent, but I think you will find that even working with half the amount of biofilm and the full amounts of the solutions will also work fairly well. If you struggle to get DNA, which I doubt will be a problem, you can add a carrier such as yeast tRNA, salmon sperm DNA or linear polyacrylamide.

Basically you will just have to try it and see, that is usually how these things have come about. You might get a methods paper out of it, if you can work it out well.


Thank you very much for your prompt reply. You say that working with the full amounts of solutions will be ok even with half the amount of biofilm used by other researchers, but what if the amount of solutions exceeds in a large extent the amount of biological material available for extraction? for example, if I work with the full volumes for 0.3 g of biofilm but in fact I obtain only a fraction of that amount, like 0.05 g (I may be exaggerating). Will DNA get degraded?

Thank you in advance.

#4 bob1

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Posted 15 November 2011 - 02:46 PM

The extraction proceedures are designed to limit damage to the DNA as much as possible. The steps can be broken down into a few simple processes - the first of which is lysis of the cells(membrane rupture - usually by detergent and/or sonication or grinding), followed by denaturation and precipitation of the proteins (salting out), and then by purification of the DNA (salt and alcohols, pH dependent to some extent).

Using 0.05 g compared to the 3 g you suggested will result in you getting some DNA out, but it will be a limited amount as there are limits to the concentration of DNA needed before the precipitation becomes inefficient. To increase the efficiency of precipitation you can add carriers, which are usually non-specific DNAs such as salmon or herring sperm DNA. You can also scale the reaction, though it gets more and more difficult to work with smaller volumes, especially if you are doing phase separations using phenol-chloroform or similar.

#5 cerbatana

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Posted 17 November 2011 - 04:05 AM

The extraction proceedures are designed to limit damage to the DNA as much as possible. The steps can be broken down into a few simple processes - the first of which is lysis of the cells(membrane rupture - usually by detergent and/or sonication or grinding), followed by denaturation and precipitation of the proteins (salting out), and then by purification of the DNA (salt and alcohols, pH dependent to some extent).

Using 0.05 g compared to the 3 g you suggested will result in you getting some DNA out, but it will be a limited amount as there are limits to the concentration of DNA needed before the precipitation becomes inefficient. To increase the efficiency of precipitation you can add carriers, which are usually non-specific DNAs such as salmon or herring sperm DNA. You can also scale the reaction, though it gets more and more difficult to work with smaller volumes, especially if you are doing phase separations using phenol-chloroform or similar.


Thank you very much bob 1, I'll follow your suggestions. Now I have one more question: when doing the cell disruption procedure, is there any specific volume of lysis buffer per gram of cells or biologic material to disrupt (when talking about grams of biofilm for example)?

Thanks

Edited by cerbatana, 17 November 2011 - 05:59 AM.


#6 bob1

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Posted 17 November 2011 - 04:20 PM

Usually enough to cover the material. I don't work with bacteria, so I am not sure on the exact procedure for them.





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