Clontech for baculovirus? NOT! NUNCA! NIEN!
greggsilk at aol.com
Mon Jun 15 16:33:01 EST 1998
>Clontech for baculovirus? NOT! NUNCA! NIEN!
>From: plxsrt at pln1.nott.ac.uk
>Date: Mon, Jun 15, 1998 04:42 EDT
>Message-id: <6m2mpg$e7j at paperboy.ccc.nottingham.ac.uk>
>I've had exactly the same experience with the Clontech vs bactobac
>Baculovirus kits. The only problems I've heard concerning the FB
>system is that you sometimes have to still do plaque assays for
>diffiult constructs. Is this your experience?
One thing about these recombination systems is that they are designed to
recombine, possibly in ways you do not wish. So the best bet is to have 5' and
3' PCR primers to check both ends of the insert in the shuttle vector construct
as well as the susequent recobination into the baculovirus....
Bactobac is cotransfection of plasmid shuttle vector into insect cells along
with linearized BEV, followed by much hoping and praying that the desired
recombination took place. I suppose it could work OK if you really experienced
with transfections, screening, and such and had all those things optimized.
With modern lipid transfection reagents, that probably won't be the limiting
FB does the recombination of BEV and shuttle in bacteria at vastly higher
efficiencies, then you miniprep the "bacmid" and transfect the goo into insect
cells. So the efficiency is high, you basically unlimited recombinant bacmid
for transfection, AND each prep is ALREADY a pure characterized clone so you
can greatly reduce the amount of plaque purification and plaque picking later.
One more hint! Don't even try to spot transfected cells in a microtiter plate.
With FB, you can count on having infected cells. Just let them grow up, split
into small flasks. If they are infected, they won't attach. Do PCR of the NPV
to check recombination.
Answer to your question, FB greatly reduces the need for plaque asssay compared
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