do nanodrop blanking solutions affect readings? - (Feb/07/2008 )
i've been using a Nanodrop to check poly(A) RNA isolation yield, and subsequent ds-cDNA synthesis yields, and the quantities are quite low - ~100ng/ul for RNA, concentrated by etoh precipitation) and ~15ng/ul cDNA, post phen/chlor and gel purification. This is not surprising given my DNA source: ants (they're very hard to homogenize). When i used the solutions supplied by the kit compaines as blanks on the nano i get very noisy curves. When i repeat with water as a blank, the curves are much more smooth (the concentrations/ratios however do not change much, although i'd say they improve a little). Of course it is best to use the storage sol'n as a blank - i'm using Ambion's The RNA Storage Solution and Qiagen's Elution Buffer, but could they affect the read? Should i even worry about the shape/aesthetics of the Nanodrop curve? Is it just because the concentrations are already at the minimum end of the Nano's range? When i get my yields up to a better efficiency will i still have this blanking problem?
Thanks for any advice!
the quiagen elution buffer is tris and EDTA so i guess it may serve as a blank.
I don't know the composition of the ambion rna storage solution. But it should serve as a blank too.
you may check it buy doing a blanck with water (not DEPC treated one as DEPC relieves alcoholic compounds when autoclaved) and then measure the OD values for either EB or Ambion' s solution. You'll be able to see if there is a deviance.
thanks dr. cat! i repeated the procedure and with higher yields, my curves and ratios look much better, even with the kit solutions as a blank. Better yield = better data. Go fig.