Baculovirus

Rick Hershberger hersh at telerama.lm.com
Wed Aug 3 10:41:06 EST 1994


In article <JEB.Harrison-030894185912 at 130.216.55.45>,
JEB.Harrison at auckland.ac.nz (Jane Harrison) wrote:

> Hi,
>                 Anyone out there have a way of getting bacuolovirus
particles out of the
> supernatant?  Spinning at 100 000xg for 60 min is not sucessful enough.

> ****************************************************************************
> Jane Harrison                     'All opinions are Mine and mine alone'
> Medical School                    "Science is like being God-
> University of Auckland                Only I have time and buget
> constraints"
> Private Bag 92019, 
> Auckland, New Zealand
> *****************************************************************************

The lab that has taught me baculovirus work (Paul Friesen, Inst. Mol.
Virol., UW-Madison) uses this procedure to prepare virions that are then
used as a source of vDNA. It may work for your purposes.

Place 6ml PBS into an SW28.1 tube.

Add 10ml of Pass 2 (high titer stock) recombinant virus supernatant.

Underlay a 0.8ml sucrose cushion (25% sucrose in NOV disruption buffer*)

Spin 60-70 minutes at 28,000 rpm at 4 degrees using SW28 rotor.

Decant supernatant immediately (should see small virus pellet).

Invert tube to drain, wipe dry.

Resuspend virus pellet in 250 microliters NOV disruption buffer.

If desired, purify vDNA by adding 1/10 vol 10% SDS, then extracting twice
with phenol/chloroform and once with chloroform and EtOH-precipitating.

*NOV disruption buffer = 10mM Tris pH 7.6 / 5mM NaCl / 10mM EDTA

Good Luck,

Rick

-- 
            RICHARD P. HERSHBERGER, PH.D.
 _  _  |_   Asst. Prof. of Biology * CARLOW COLLEGE
|  |_| | |  3333 Fifth Ave. * Pittsburgh PA 15213
   |        hersh at telerama.lm.com



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