Why is my second DNA elution have lower 260/280 ratio?

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Why is my second DNA elution have lower 260/280 ratio?

Postby chunchunmaru » Apr 16 2017 9:25 am

Hi,

Can anyone explain this: how/why does a second elution of DNA have a lower 260/280 ratio (and 260/230 too!)? Also, why is the first elution really bad; smears are very prevalent. The 2nd elution always has nice and high MW bands with very little smears. But, the problem is that the 2nd elution has low ratios: <1.5 for both ratios. And what puzzles me more is that 1st elu looks horrible in gel but really pretty in spectro (~1.8 and 2.0 - 2.2), plus high DNA quantity.

I am using a column-based, commercial extraction kit and I will be using the DNA for SNP genotyping using Sequenom. Nanodrop for spectro measurement. Any ideas?

Thanks!
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Re: Why is my second DNA elution have lower 260/280 ratio?

Postby 29yrsExperience » Aug 29 2017 7:39 am

I assume you are purifying genomic DNA from cells/tissues. Genomic DNA always looks a bit smeary (it is fragmented, randomly, by the isolation process) but as long as most of it is high MW is should be OK. It will smear a lot if you load too much on a gel with a percent agarose not optimal for its size. If it smears in the lower MW range you might suspect there is RNA that is not being removed by the RNAse in the kit (kit too old?). I'd expect the 260/280 ratio to decline a bit in a second elution- if more protein contaminants are going to be washed out it would most likely happen in later elutions. As for the 260/230 ratio, this is highly influenced by the kind of chaotropic salt that is used in the purification (usually guanidine HCl or guanidinium thiocyanate). These salts can be carried over in your eluate, and the latter has significant absorbance at less than 240 nm. (Usually the carryover does not affect downstream applications.) Please read your kit instructions thoroughly; or for more information look on-line for the Macherey-Nagel Nucleospin gel and PCR clean-up kit manual-it has a really good explanation of the chaotropic salt issue (and I credit it for my knowledge.)
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