SOS: DNA purity problems - Low A260/230 of gel extracted DNA samples (Jan/14/2008 )

Hi all

Recently I've faced a DNA gel extraction problem. Usually I fully digest 2-3ug of plasmid DNA (for further cloning) with sticky-end RE. Then load each sample into 2-3 wells of 1% agarose/TAE gel, then the samples are separated and dyed with EtBr. Bands of interest are excised from gel using a UV-light table and then I proceeded to gel extraction using any column kit (Zymo, Qiagen, ABgene... you name it) following manufacturer's instructions. Usually the DNA yield overall is high (100-150ng/ul) with A280/260 1.7-2.2, however ratios A260/230 almost always are extremely low (>0.1). Thus my question is what might cause such low A260/230 ratio? How do I prevent this from happening in the future?

Can anyone advice me on the subject?
Appreciate your help.

The reason is a guanidine isothiocyanate contamination. If you need a better ratio, you have to precipitate the DNA. But the manual of your extraction kit should tell you more.


For normal cloning we never had a problem with this low ratio.

or major contamination is obtained by bad drying of the column.
ex : in a column kit it's written spin 1' at top speed and spin 2' at top spin to finish dry. You can directly add water to elute DNA. That's false. You need at least 37 ° 10' or 55+5' on a thermo block to efficiently remove the remaining wasdh solution.
230 ratio will reduce strongly.