Low A260/A280 ratio after gel purification using Promega PCR Clean-up System - (Sep/29/2011 )

Hi

I have just completed gel purification using the Promega Wizard SV Gel and PCR Clean-Up System kit for the gel slices of my PCR products. Prior to this step, I amplified the PCR products using gel electrophoresis in 1% agarose gel and obtained relatively sharp bands (but with highly visible primer dimer). There seemed to be no obvious problems prior to gel purification until I observed that the values for A260/A280 ratio of the products to be much lower than 1.8 after doing Nanodrop of the purified PCR products. Therefore, I think that any problems that should be present would only have surfaced during the gel purification process. However, I am stucked in a rut as I have repeated the entire procedure twice and still could not find the root of the problem.

Can anyone enlighten me?

Hi badonkadonk. The first question I'd ask is whatwere the actual A260 and A280 values? A low A260/A280 ratio could be due to a high number in the denominator (A280) -OR- a relatively low number in the numerator (A260). If there was not much DNA in the sample then the A260 may not be much above background. Did you get a good PCR yield? Intense band? Did you expect a lot of DNA?

Please feel free to enlist Promega Technical Services to help troubleshoot any issues, in addition to our colleauges in this forum. You can reach us by e-mail, chat or phone through www.promega.com.

I'm not sure how ethanol contamination affects absorbance, but our lab was having some problems with this kit. We increased the final spin from 1 min to 5 min or more (and put the column into a completely dry tube for this) and that resolved the problem for us. Don't know if that helps you though.