Begining with Baculovirus

Absolut Queer janeh at ihug.co.nz
Sun Jun 23 05:12:48 EST 1996


mlush at hgmp.mrc.ac.uk (Mr. M.J. Lush) wrote:

>In article <jpl-180696150831 at mac-jpl.ibcp.fr>, jpl at ibcp.fr <jpl at ibcp.fr> wrote:
>>
>>	Hi! I will soon try to express the cDNA coding for my protein in a
>>Baculovirus expression system. There are several kits and several vectors
>>available to start such a job. The Novagen one is attractive according to
>>is lower price. Let me know your experience with it or give me any valuable
>>advice on how to begin with Baculovirus. Thanks for all.

>	The girl on the next bench is working with Baculovirus...
>the main problem turned out to be that you need to use a cell line
>with a low passarge (Sp?) number.   This fact was not mentioned
>in any of the protocols and only transpired from conversation 
>with the tech support (eventually!).
>-- 
>Michael

The Pharmingen (no affiliation) manual, and the Baculovirus manual you
can order from Sigma (no affiliation) both make mention of this fact,
albeit hidden well within the text.

Don't grow any cultures for more than 3 months, freeze down a whole
bundle of cells (like 30-40 vials) as soon as you can form the first
cells you receive from the kits.  

Another hint, pehaps not accentuaed as well as it could be in tech
manuals-Make sure what you are using is a clonal virus, use  a serial
diltuion analysis, and take a clonal virus from  <5/36 wells infected
at high (10E-6 or greater) dilution.  Or use plaque assays, and repeat
three times or more to ensure a clone.  It saves heartache in the long
term if time is taken at this point.

If possible have a check for expression, and test that the virus is
expressing the protein, it is possible to get a non expressing clone
(rare, but its a bitch when you do-wrecked my Masters with a non
expressing clone).  

Otherwise, have fun. It is easy once you get it going.

JAne

Jane Harrison
Absolut queer

"Science is like being God
Only I have time and budget constraints"




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