virus purification needed

M K Bennett pamkb at mail.bris.ac.uk
Tue Feb 28 04:29:52 EST 1995


WAS (SHOREWA at UCBEH.SAN.UC.EDU) wrote:
: 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.  Another question I have is what would be the difference
: between Proteinase K and Pronase ?

: Thanks for any help 

OK, this is what I do for a CRUDE herpes simplex DNA prep.

    1. 	Infect your cells, wait for CPE, shake the cells off of the flask
    2. 	Spin down the cells in a refrigerated Centrifuge 3k, 10min.
    3. 	Decant the supernatant into fresh tubes and spin this at 13k for
	1/2hour. This brings down some viral particles in the supernatant.
	I know that this shouldn't really work (g forces and all that) but
 	it does.
    4. 	Gently resuspend the cell pellet in 5.0ml 0.5% NP-40 in PBS (play 
	with the detergent concentration, 0.5% works great with Vero cells).
    5. 	Spin down the cellular debris 2k 5-10min.
    6.	Remove the supernatant and save in a fresh tube.
    7. 	Resuspend the viral pellet from step 3 in 2.0ml of NP-40/PBS and
	combine with the supernatant from step 6. 
    8.  For each 5ml of extract add 25ul RNaseA (10mg/ml), 100ul
	proteinase K (100mgs/ml), 250ul of 20% SDS and incubate the mixture
	at 65C for 1 hour.

then phenol chloroform extract the DNA from the extraction.  You will get
some contamination with Genomic DNA from the cell line but there should be
enough viral DNA for you to work with.  An alternative is to only use the
viral DNA from step 3.

mike
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Mike Bennett D.Phil		|Scientists have odious manners, except when
Dept Pathology and Microbiology,|you prop up their theory; then you can borrow
University of Bristol, UK	|money from them--Mark Twain 1917 
m.k.bennett at bristol.ac.uk	|
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