We isolated some Acanthamoeba cysts after filtering of water samples, we need its DNA for PCR but there is some difficulties for breaking the cysts wall after using all physical & chemical usual methods.
Hi there I have worked with Acanthanoeba in a drug screening initiative in an effort to destroy the trophozoite form and prevent encystation. I therefore appreciate the refractory nature of the cyst and associated difficulties with harvesting DNA. Have you tried DNA extraction with chelex resin. Find attached 2 papers describing the use of such resin and a 3rd paper with an alternative. I appreciate However that you claim to have exhausted all conventional methods which might include this method. If so I will add 1 more thing. You talk about harvesting cysts presumably from a wild type environment ? If you can dispense with wild type sampling it might be more productive to culture up trophozoite cells and induce encystation with hypertonic salt media and then selectively enrich for cysts by ablating trophozoite with SDS followed by filtration to remove encysted clumps which are highly refractory to treatment. This should result in a suspension consisting of single cell cysts. Subject this to chelex resin purification although you might need to start with a lot of starting material as you could lose a large proportion of your starting titre in the absence of dispersing cell clumps. In essence if you are directly filtering of cysts a paucity of material could be your problem. This is because DNA is not liberated from enough cysts by virtue of their mechanical resistance and consequently you need to start with a high cyst titre to liberate enough raw DNA for down stream purification purposes. I hope this is of some help. Good luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020456/
https://lra.le.ac.uk/bitstream/2381/9213/2/Chelex%20manuscriptJCMmodclean2.pdf
http://onlinelibrary.wiley.com/store/10.1111/j.1365-2672.2010.04708.x/asset/j.1365-2672.2010.04708.x.pdf;jsessionid=70C7B0F7130DE3C7082B90DD23FC689D.f01t04?v=1&t=icc7y3q1&s=942410bf62b6f709c868826650768ab925636113
Dear Laurence Stuart Hall
Thank you very much for valuable response. I would do some experience as you wrote. hope to work well. I should write the results for you.
Dear Laurence Stuart Hall
Hi
Would you please send me the full paper of above articles number 2,3. I could not access to their sites. Thank you. [email protected]
Hi Abbas
I have attached the pdf. files of those chelex resin protocols
Hi Abbas,
i had some problems with my DNA and the extraction method but i was working on Cryptosporidium not Acanthamoeba, anyway i use 3 -5 times freezing -thawing( boiling) in -196 and 100 celsius degree and i used the traditional fenol-chloroform, but also Qiagen kit it was helpful, hope u,ll find ur way.
Good luck !!!
Regards
Nima
Dear Dr Eskanderian.
Salam
The best method is cultured the cyst in bactoagar plate nutrinet with Eshershia and then extracted DNA from cyst and throphozite. This cultur medium very easy and cheep.
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