baculovirus kit opinions needed
mskidmo1 at san.rr.com
Tue Mar 3 03:37:11 EST 1998
I've used Pharmingen BaculoGold in the past and had no problems with it. But
if you use it don't use that silly CaPO4 included. Buy CellFectin from Gibco
(It's lipofection for insect cells). 100 - 1000x higher tranfection of your
DNA. I was initially excited to try the Gibco BRL BacHis system but a collegue
spent 3 months trying to get recombinants. They finally attributed it to poor
cells after I gave them a fresh log culture that was low passage #, I am very
suspect of this explanation. they eventually got the construct when I would
have had it in 2 1/2 weeks with BaculoGold. The only real advantage it confers
is blue/ white screening to help you pick plaques (nice for begginers). But if
you just follow the protocol in Luckow's Manual of Baculo Tecniques on Phenol
Red staining of plaques, blue/white becomes less important especially when
you are going to have 90% + recombinants from a lethal deletion. Against my
advice, a lab partner is doing BacHis right now (ligation into transfer vector
is currently at 14 º) Ask me in two weeks how it went.
I have been wanting to try the new Novagen Bac3000 which has deletions of the
AcMNPV cys-protease and chitinase and lead to less cell lysis and recombinant
proteolysis (they say). Also since the genome is smaller your insert can be bigger.
As you compare the kits you may want to check the Invitrogen Baculo User Web
site discussion board. <http://www.invitrogen.com> this is a common question
and the archives are a great source of all kinds of info for beginners and
experienced users as well.
Whatever you choose, don't buy into the sales pitch that a kit doesn't need
plaque purification. I spoke with Pharmingen, Invitogen and GibcoBRL and they
all do 1-2 rounds of plaque purification despite the fact that the sales
literature syas you don't need to. If their own tech support and custom
expression people still do it I think that's pretty good advice to not skip
that step. It only takes another week and you can wait and do it after you
have confirmed that something expresses well.
The first BacHis user lab I mentioned skipped this step and after 3 months of
high level expression the viral stocks were taken over by either wt or
deletion mutants and they had to start all over again. Even the original
stocks from the first few passages went bad after a few rounds of scaling up.
Michael Skidmore, grad student W: (619) 784-9866
The Scripps Research Institute H: (619) 587-4885
Molecular Biology, MB13 <http://www.scripps.edu/~skidmore/>
10550 N. Torrey Pines Rd
La Jolla, CA 92037
"Standard orbit, Mr. Sulu."
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