Q: Transfection control plasmid

Eugene ronlab at uic.edu
Wed Mar 4 11:05:58 EST 1998


Hi!

    We found that presence or absence of an insert (promoter is there in either case)
makes a noticeable difference for expression of another gene (e.g. marker) within the
same construct.
    Also, if an insert substantially changes your plasmid size and you are using
stable transfection, it may alter the predominant structure of the integrated
construct. If you are using retroviruses, it may affect the packaging efficiency, the
titer and, consequently, copy number of integrated proviruses per cell. If you are
using episomes, a big insert may affect their stability.
    My personal preference is using a biologically inert insert of similar size, but I
am not sure whether your truncated variant is the one (read-through, etc.). I guess
you are the only one who can judge what is a " biologically inert" insert for your
studies.
    By the way, for the purposes of publication you will easily get away with an
"empty vector" control.

Eugene Kandel
U09577 at uic.edu

>
>
> Wolfgang Schechinger wrote:
>
> > Hi all,
> >
> > What would you recommend as better control plasmid
> >
> > 1) the vector without insert
>
> > 2) a mutation with the same sequence but a deletion of one base
> > right after the ATG (so the message is transcribed, but translation
> > soon will be terminated due to running into a stop) - I wouldn't have
> > to prepare it, just got this one accidentally. #:-(((
> >
> > What's your opinion on that?
> >
> > Wolfgang
> > -------
> > !Junk mail is *not* appreciated!
> > SPAMs will be answered automatically by sending my 128MByte virtual memory file.
> > This is *NO* joke.






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