I have a mixture of Archaea and I would like to disrupt their cell walls.
Lisozyme and proteinase K are not working.
You can increase the incubation time after the treatment and if after that also not working then you can go for sonication.
Hello there,
Archaeal cell wall composition suggests that you shoud be able to disrupt the cells with proteinase K and mix of detergents. Perhaps the detergents you are going to use are more critical.
In response to your question; It is possible to disrupt archaeal cells with proteinase K in lysis buffer containing Triton X-100, SDS, and N-lauryl sarcosine. I was using that method as a standard step in genomic DNA extraction from P. furiosus.
Hope this helps
Sj
is enzymatic disruption obligatory? why not SDS or bitbitter or freezing-warming?
Lysozyme will not work due to the cell wall properties of Archaea. We recently switched from proteinase K to 0.1N HCL to permeabilize archaeal cells for CARD-FISH analyses. This seems to work well, so maybe this could be a solutions for you too. If you do a websearch in this respect you will find some papers e.g. http://www.ncbi.nlm.nih.gov/pubmed/22159570. Alternatively check your proteinase K there is different qualities that might yield different results.
Can you describe the composition of the Archaea cell walls you want to disrupt?
I have found that cell wall substance that need to be disrupted are Pseudomurein and glycoproteins. See the article by Ganesh Ram R. Visweswaran,1, 2 Bauke W. Dijkstra,2 and Jan Kok1 "Two Major Archaeal Pseudomurein Endoisopeptidases:
PeiW and PeiP" at Hindawi Publishing Corporation Archaea Volume 2010, Article ID 480492, 4 pages. And the attached article from where I only have the attached page.
Use a motar pestle, add some liquid nitrogen, and crush away.
Follow this with standard dna extraction kits or phenol chloroform.
This would work for most standard molecular biology based applications downstream except for large BAC libraries.
In case you need high molecular weight methods and want to use only high molecular weight DNA you might have to use an in gel lysis method using blocks like in preperation for PFGE gels.
Archaean cell walls do not contain peptidoglycans, so lysozyme is useless for disrupting the membrane.
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