?plasmid flourometry standard

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Fri Feb 3 12:01:18 EST 1995


In article <Pine.A32.3.91c.950131204730.112395C-100000-100000 at homer07.u.washington.edu>, Robert Pierce <rpierce at u.washington.edu> writes:
>I'd like to quantify a plasmid flourometry for some 
>dilution experiments and have been told that I can't use the usual linear 
>calf thymus standard.  Is it true that flourometry is sensitive to 
>DNA configuration?  Are quantitative plasmid standards available?  Thanks 
>for any info. 
>
Yes, the dye used to measure DNA concentration in a fluorimeter is said to
be sensitive to DNA GC-content and to the configuration. So, if you need
absolute amount of plasmid DNA, you may have to buy a supercoiled standard.
However, you may also prepare it yourself by isolating a relatively large
amount of the vector under your conditions ( strain, media etc ), quantify
it by UV absorbtion spectrometry and use as a standard. Caution: don't
use CsCl centrifugation, column chromatography is preferable, since it
will eliminate RNA and does not use ethidium bromide ( i.e. Qiagen etc )
Standard disclaimer applies.

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich

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