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I am encountering a problem in an apparently easy step: DNA purification from agarose gel.
I depart from a 8,3 kb plasmid. Then, I cut aproximately 3 ug of that plasmid at a single site, thus linearizing it. After that, I perform a defosforilation reaction using CIP (Calf inestinal Alkaline Fosfatase). The point of this process is to obtain a negative control for ligation (a plasmid that cannot religate). Immediately after defosforilation, I prepare a 1% agarose gel and run the complete 3 ug of DNA during 60 minutes. Then, I cut the bands that supposedly contain the linearized and defosforilated DNA and proceed to purify it through centrifugation using Invitrogen's latest PureLink Quick Gel extraction kit. I strictily follow the protocol, but when I finally measure the concentration of purified DNA using a Nanodrop ND1000 I experience some kind of contamination that produces a peak of absorbance at about 230 nm. After carefully reading the protocol, I think it could be caused by Guanidine Isothiocyanate, a caotropic salt contained in the buffer L3 (which is used to solubilize the gel band when excised). Following the recommendations suggested in the troubleshooting section of the kit's users manual, I repeated the purification adding a second wash with the buffer W1 (a buffer that contains ethanol and is used after the gel is solubilized), with no positive result. The peak at 230 nm reapears.
Has someone experienced this type of problem before? How can I overcome it? Could contaminants such as guanidine Isothiocyanate affect a downstream ligation reaction? Every help will be appreciated.
Edited by DH5a, 18 March 2009 - 01:52 PM.
