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Eliminate bacteria(l DNA) from hypochlorite-treated nematode eggs


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#1 Aucklander

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Posted 08 September 2010 - 09:41 PM

Hi,

I hope this is the right sub-forum for this question. I'm currently trying to extract DNA from bactreia-eating nematodes for sequencing purposes. My take is to use hypochlorite to digest adults and bacterial contamination, and extract pure nematode DNA from the eggs. So far results have been mixed; adult cuticles remain, and I'm worried that they might contain residual degraded DNA or that bacterial DNA might remain in the solution. Is there a way to separate out/purify the eggs after hypochlorite treatment? Or am I just worrying to much? As far as I have seen no paper dealing with sequencing or array-work in nematodes has described this process in detail.

The answer to this question has eluded me for some time now, helpful input is much appreciated!

#2 Adrian K

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Posted 09 September 2010 - 03:55 AM

hi,

I wasn't in your field of study but since you only wanted the DNA of the nematode, is it possible after you had remove the bacteria, you passage the nematode for 2 or 3 generations (by feeding your nematode with some other thing else) with antibiotics to ensure the total removal of bacteria?

and since you are able to remove the eggs, possible to add DNAse? (with assumption your dnase will not enter through the nematode's egg shell)?

Just my 2 cent.
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#3 gebirgsziege

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Posted 10 September 2010 - 12:47 AM

Hypercloride should remove DNA contamination on the outside; but are you sure that there are no bacteria INSIDE the nematode eggs, thinking of some mutualistic symbionts???
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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