RNA quantification & Northerns

Rosemary Hoffman rth at anat.UMSMED.EDU
Thu Apr 15 00:00:19 EST 1993


In article <93104.080826SSD4 at psuvm.psu.edu> <SSD4 at psuvm.psu.edu> writes:
>Does anyone out there have the secret for obtaining repeatability in
>loading northerns?  Currently I am using A260 to quantify the RNA in my
>samples and then determining the volume required for 10 ug.
>....[deleted]
>Is there a better method than absorbance @ 260 for quantifying RNA or is
>there a possible problem in my sample prep.
>
>Fishing for answers / suggestions
>
>Shawn S. Donkin

Shawn,
Are you reading the OD only at 260?  Try reading at 230, 260, and 280 nm
and checking the OD ratios.  Residual organics can falsely raise the 260
but they will be picked up by a high A230.  Protein contamination will
be seen in a high 280.  I don't remember the exact ratios but you can
find them in Methods in Enzymology (an old volume) or probably in "The
Big Red Book" or Maniatis.

What kind of OD readings are you getting?  If they're low (perhaps <.1
or 0.2) you may just be looking for too much accuracy from an inexact
reading.

Rosemary (world's oldest biochem grad student - the Meth.Enzymol reference
should tell you THAT!)



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