precise quantification of genomic DNA

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Dec 20 10:07:18 EST 1994


Brian C. Freeman writes:

> Need Help: I have problem in quantification of genomic DNA from different
> cell lines. The DNA samples have been treated with RNase A,
> phenol-chloroform, and the OD260/280 are higher than 1.8. Loading the same
> amount of DNA(based on OD260 measuring)  from different samples in agarose
> gel, however, gives a significantly different fluorecent intensity. Should
> I trust the result from OD measuring or from agarose gel?  

1. RNAse converts RNA to small oligos and nucleotides.  These absorb light
   even more strongly than the RNA did, and with the same spectrum as RNA
   or DNA.  So unless you do something to physically remove the RNA or the
   digestion products, your OD will still be inflated.  Typically people
   rely on ethanol precipitation for removal of the digestion products,
   but this often leaves a considerable residue of RNA oligos.

2. Also measure the OD 230.  DNA or RNA peak around 260 and come back
   to a minimum absorbance around 230 approximately the same as the 
   OD 280.  Some small molecules (EDTA in particular) give an OD 260/
   280 similar to DNA, but just keep going up at shorter wavelengths.
   If you don't actually have a peak at 260, your OD 260 is meaningless.

3. If the spectrum is approximately like DNA, then an OD 260/280 as high
   as 2.0 is probably not a serious problem.  It means the DNA is not
   totally pure, but usually it's pure enough.

4. When you run the gel, make sure you use a quantitative standard, say 
   some uncut lambda DNA.  If the samples aren't the same as each other,
   then they may be grossly below the concentration predicted from the
   OD.

5. A common problem with high molecular DNA (say after an ethanol precip.)
   is to not get it completely redissolved.  Then each time you take a
   few ul as a sample, you get a grossly different amount.

6. Other than problem 5 above, and assuming that you have a reliable
   quantitative standard, the gel is more reliable than the OD.  It
   is only limited by your ability to quantitate the fluorescence, which
   is mainly limited by the number of standards that you load.  If you
   are quantitating by eye, however, you probably can't do better than
   a factor of 2-3.  The OD is theoretically capable of quantitating to the
   3rd decimal place, but this is only relevant if you have other information
   that the DNA is pure.  For impure DNA, the OD is mainly useful to
   warn you of what kinds of contaminants are present, and approximately
   how much.

Hope this helps

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu
 



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