Clontech for baculovirus? NOT! NUNCA! NIEN!
blaesse at biochem.mpg.de
Tue Jun 16 03:53:59 EST 1998
I want to add a comment to this discussion:
> Sorry if there was any confusion in the previous posting. I had not expected to
> get this involved, so I just went downstairs for the Bac-to-Bac manual.
> Clontech makes BacPac.
> Life Technologies (GibcoBRL) makes Bac-to-Bac which uses the pFastBac (FB)
> vector as the donor plasmid. I did this 18 months ago, and even then all the
> "bac-this" and "bac-that" got confusing at times.
> Since I have this stuff in front of me.....
> Bac-to-Bac (FastBac) (FB) (LifeTech) (GibcoBRL) has:
> High efficiency recombination in a special Ecoli host strain.
> Strong selectable ANTBIOTIC markers for recombinant virus, not wussy
> Screen and grow bacmid in Ecoli before transfection.
> No nonrecombinant virus background, avoids marathons of plaque purification.
I used this system. As I did not know how well it works I have done one
round of plaque purification. I had some strange results: from some bacmid
preparations I got no virus clone which expressed the desired protein.
Ok, this is easy to explain: the blue-white selection for the recombinant
did not work. But from some bacmid preparations I got a few virus clones
which expressed the desired protein and a few which did not. Although I
checked the E. coli clones which I used for the bacmid preparation again
on a plate if they stayed white.
> Efficient transfections using
> MOST important, the limiting reagent is not obscenely expensive linear virus
> (like certain other companies). The imiting reagent is the host Ecoli cells
> which were $15 a tube and work very well.
> Is expression cloning simple? No, it's still a big committment and it takes
> very savvy planning, but there is a reasonable chance of success.
> Your mileage may vary ;)
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