Problem with DNA purification from agarose gel - (Mar/18/2009 )

Hi to everyone. First of all, I'd like to highlight the fact that this is my very first post and also I am a very inexperienced researcher, so try to be comprehensive.

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.

Hey

What do you elute the DNA in? Does that absorb at 230nm? Did you record an absorbance spectra for that?

Frankly I am not sure if it would affect the ligation but you always have an option of avoiding the kit and precipitating the DNA down.

Best
TC

T C on Mar 18 2009, 07:41 PM said:

Thanks for your time, TC. I elute the DNA in nuclease-free Milli-Q water, avoiding the elution buffer provided with the kit (which contains Tris-HCL). I preheat the water to 65 ºC before eluting (suggested by a coleague), and apply 30 ul to the center of the purification column. Frankly, I am kind of annoyed with this contamination. I hope this problem will be solved soon.

DH5a

Hey

That seems fine.

Just precipitate the DNA then. Add one volume Sodium acetate pH 5.4 followed by addition of 2.5 volume ethanol. Incubate at -20 for 2 hrs (you can even keep it overnight). Next morning spin it down, give washes with 70% ethanol and dry. Resuspend and use.

Right now, do proceed with the ligation with whatever you have in hand, you may get some colonies. After elution I just run 3 ul DNA on a gel, get a rough estimate of the concentration by comparing with the ladder, set up a ligation, transform and I get clones. Mine is a molecular biology lab and we do a lot of cloningso we try to save time by not finding theexact concentration. Wrong practise but it works.

Hope it helps.

Best
TC

DH5a on Mar 19 2009, 04:14 PM said: