Transfection question

Martin D. Leach leach at bu.edu
Wed Apr 5 13:33:55 EST 1995


In article <3ltvql$85b at biovax.biobase.dk>, sjn at biobase.dk (Soeren Jensby
Nielsen) wrote:

> Hi
> 
> In most of the articles that I have read concerning deletion analyses of
> promoters/enhancers the authors transfect a fixed AMOUNT of reporter plasmid
> together with an internal transfection standard. Now if you're interested in
> examining say 2 kb of promoter/enhancer in this way and your full-length 
> plasmid is say in the order of 6 kb, then you actually transfect at least 30%
> more moles of plasmid in the case of the fully deleted fragment if you stick
> to a fixed AMOUNT of reporter plasmid. Now is this not conceptually wrong?
> 
> Soeren

Results are normalised to the internal standard.

1. If too much internal standard is used then you have a massive signal.
You want the signal measured to be in the linear range.

2. Again, too much internal standard may cause some interference..e.g.
some viral promoters can compete for factors from the promoter you are
studying..and diminish/interfere with the promoter to be studied.

Above is not conceptually wrong...you just have to figure the role and use
of the internal standard.

Martin

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