DNA OD readings help

Anton Scott Goustin asg at cmb.biosci.wayne.edu
Sun Nov 26 15:29:39 EST 1995


tcowan at morgan.ucs.mun.ca (Theresa Cowan) wrote:
>After PCR anplification, chloroform extraction and column purification.  
>The 260/280 readings for my samples are between 2 and 4 (14 samples).  
>I've asked around about this, only to get two different opinions.  1)  
>The samples are very pure.  2)  The samples are contaminated. 
>If I chloroform extract and ethanol/isopropanol extract my PCR products, 
>the 260/280 readings are between 1.7 and 2.
>Any suggestions?
>
DNA should not exhibit 260/280 ratios greater than 2.0; the readings 
between 1.7 and 2.0 seem normal.  (The precise 260/280 ratios of pure
DNA depend upon its base composition.  For example, A-rich DNA should
have the highest 260/280 ratio, reflecting the inherent absorption
spectrum of adenosine (max at 257 nm)).  I suspect that, in addition
to DNA in the first samples (ratios >2.0), there is a substance, per-
haps leached off the columns, which absorbs highly in the 260 nm
area, boosting the observed 260/280 ratios.  If you load 0.01 OD 260
units of DNA from each method on a 1% agarose gel, separate, and stain
with EtBr (should be ~400 ng of DNA), how do the two bands compare in
brightness?




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