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PCR from roots


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2 replies to this topic

#1 scgradstudent

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Posted 13 July 2009 - 01:12 PM

Hi,

I know the title sounds a bit crazy, but just bear with me for a second. My advisor is wanting me to take roots samples (1mm in length) and place it in a PCR tube and try to amplify DNA from the sample. We are trying to amplify 16SrRNA from the bacteria associated with the roots without doing DNA extraction. We are trying this just to see if it would work. I've tried to look up to see if anyone has tried this, but I have not found any literature. Has anyone tried a protocol like this?

To me, this would be similar to taking a bacteria colony and smearing it on the bottom and adding the PCR Master mix. I'm just wondering if there is anything different I would need to add to the PCR mix, change to the protocol, or possible treatment of samples? Thanks!

#2 ivanbio

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Posted 14 July 2009 - 03:58 PM

If you are trying to detect the bacteria associate with the roots, then your issue is a little more complicated than simply running a qPCR reaction directly from root tissues. For example how do you know that the bacteria left over after you clean the root is associated with the root and not just bacteria that happen to be in the soil? Also, if you leave the root in the qPCR tube, root cells will likely break open during PCR, which will lead to any bacteria inside the root to be amplified too.

These issues aside, you may be able to get this to work, but just like bacterial colony PCR it is not going to be very reproducible.

Good luck

Ivan
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#3 gebirgsziege

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Posted 14 July 2009 - 10:33 PM

There is a paper with a method like this for published for mycorrhiza (rapidPCR or something like this), but I am sorry I do not remember it in detail. Maybe you can find it.

If your attempt works might be mainly depending on the nature of your root samples and the amount of bacteria of interest present. And the puritiy of your sample, like how many other bacteria are present in your sample (Remember you probably will not get a "sterile" root with only your bacteria)

Nevertheless I would give it a try, with an extended 96 (94) C step in the beginning, like 5min.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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