MadSci Network: Molecular Biology

Re: why acidic buffer condition for RNA isolation and basic buffer for DNA?

Date: Thu Jul 27 19:29:12 2000
Posted By: Sean Blasdall, Staff, Civil & Environmental Eng, University of NSW
Area of science: Molecular Biology
ID: 964678288.Mb

its true that the buffers are acidic for RNA work and slightly basic 
(usually around pH 8)for DNA extractions.  If you place DNA in a strong 
bacic solution the bonds between the strands will be cleaved and the 2 
stands will not be able to re join.  RNA can survive and contaminate when 
preforming a DNA extraction, unless specific steps are used to remove the 
RNA ie using RNase.  The acidic conditions for RNA extractions is due to a 
number of problems when dealing with RNA.  DNA is a major contaminant which 
needs to be removed as it can interfer with RT-PCR etc.  Also RNase has 
very little activity in acidic condition and is de-activated by guanidinium 
thiocyanate. Often the RNA extraction buffers are 5M G.thio and pH 4.8ish.

hope that helps

Admin note:
Another reason for the disparity in nucleic acid extraction buffers is tied to 
the phenol extractions commonly used to purify the nucleic acids away from 
cellular proteins: DNA is soluble in even mildly acidic phenol while RNA is not; 
so a good way to purify RNA's away from genomic DNA is to perform an acidic 
phenol extraction.  Since phenol is itself a buffer, these extraction work best 
if the sample buffer is also acidic.  Conversely, if DNA is being isolated, any 
acidity present during phenol extraction could greatly reduce yield.

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