RNA quantification & Northerns

Daniel Meier dmeier at post.its.mcw.edu
Wed Apr 14 08:57:02 EST 1993

SSD4 at psuvm.psu.edu wrote:
: 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.  After drying
: in a speed vac I dissolve the RNA in 2.2 ul of 3X MOPS/EDTA before adding
: 4.8 ul of formaldehyde/formamide mixture and heating to denature the RNA.
: Regardless of the time and attention spent in sample preparation I
: invariably end up with inequal loading (based on staining with ethidium).
: 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


  I found that to get equal loading you should have 260/280 ratios greater
than 1.7, and your 325 nm reading should be less than 0.006.  If your 
325 nm reading is relatively high, then you have debris in your sample. 
Routinely, whenever we quantitate RNA using UV absorbtion, we always
include a 5 min spin (10,000 x g) to pellet debris.  The clear supernatant
is then transferred to a fresh tube.  Don't hesitate to leave 10
microliters behind with the debris.  Also check if your cuvettes are clean
and not scratched.

  Good Luck,

  Dan Meier
  dmeier at post.its.mcw.edu

P.S.  email me if you have any other questions.

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