Endofree Maxiprep and TOP10 cells

Gumby2AL esh at nospam.uab.edu
Fri Jul 14 11:18:06 EST 2000


As a whole the Qiagen kits work great, however we have gotten poor yields
with certain plasmids and cell types.  The following is a list of problems
and solutions that we have come up with in our lab to increase the quality
and quantity of our plasmid DNA:

1.  If you have a low copy number plasmid, you will get a lower DNA to
protein ratio.
     (Either subclone your DNA into a better vector or try chloramphenicol
amplification)

2.  If do not get all of your DNA into solution, you will get a lower DNA
to protein ratio.  (We use
     TE pH 8.0, since the higher pH seems to help the DNA get into
solution, and we heat the DNA
     in a water bath.  Furthermore, do not over dry your DNA pellet)

3.  If you have a high copy number plasmid, you may be overloading the
column.

4.  If your buffers are old, you may need to add additional RNase.

Hope this helps.  I know that a lot of this information is rather basic,
however often it is easiest to overlook the simplest of solutions.


Bertrand Collet wrote:

> Hi everybody,
>
> I am having some problem of DNA quality with the Endofree Maxiprep
> Plasmid purification kit: A260/A280 ratio are never above 1.4-1.5 when
> this protocole is supposed to give ratios above 1.8. I tried to reduce
> the volume of the starting culture, use twice reagent for the alkaline
> lysis ... there is no way to improve the DNA quality. Are the TOP 10
> cells in cause (high carbohydrate content ...) ? Is it worth changing
> bacteria strain ? Is there anybody who had this problem with TOP10
> strain ?
> I do need top quality DNA for transfection purpose.
>
> Thanks for your help,
>
> Dr Bertrand Collet
> University of Aberdeen
> Department of Zoology
> Fish Immunology Group
> Tillydrone avenue
> Aberdeen AB24 2TZ
> Scotland
> Phone: (+44) (0) 1224 273 796
> Fax: (+44) (0) 1224 272 396






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