virus purification needed

RYBICKI, ED ED at molbiol.uct.ac.za
Tue Feb 28 08:06:11 EST 1995


> I have been assigned a task to isolate the DNA from a circovrus.  
I have> tried a few protocols  but nothing has seemed to work.  I 
would like some> information on DNA isolation techniques for ssDNA 
and possibly dsDNA from> virus.  Basically what I have done so far 
is to isolate the avian genomic> DNA in hopes that the smaller  2kb 
viral genome comes with it. I have had> no success as yet.  I posted 
this query to molbio.methods-reagents and> came up with a Hirt 
extraction.  Haven't tries the Hirt yet,  but will> this morning.  

I would suggest at this point - IF circovirses replicate like 
geminiviruses, which I am sure they do - that you attempt a 
geminivirus-derived replicative form DNA extraction protocol.  With 
geminiviruses you can isolate total host genomic NDA, then pretend 
you're doing an Ish-Horowicz-Burke type plasmid DNA purification 
(alkaline denaturation, CsCl fractionation).  You get a plasmid-like 
cccDNA representing replicative form minichromosome-like DNA, much 
easier than by purifying circular ssDNA from particles in many 
cases.  Try Hughes et al. (1993), Arch Virol 132: 171-182, and 
references therein.

Good luck,



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