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Low A260/A280 ratio after gel purification using Promega PCR Clean-up System

Promega PCR gel purification A260/A280 ratio

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#1 badonkadonk

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Posted 28 September 2011 - 11:54 PM

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?

#2 Carl@Promega

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Posted 29 September 2011 - 08:13 AM

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.

#3 leelee

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Posted 29 September 2011 - 06:28 PM

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.





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