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?
