gel purification of PCR product - (Sep/05/2006 )
I had a problem when try to purify PCR product from gel.I'm using Qiagen gel extraction kit to purify DNA fragment from gel.I got a very shap and distinct band from PCR amplification in the agarose gel after the electrophoresis.I cut the gel and purified with Qiagen gel extraction kit.When i run the purified product on gel, i can get back the expected size of PCR product (~700 bp).However when I use the purified PCR product as a hybridization probe in the Southern blot, I noticed a extra band more than 700 bp in the positive control (PCR product as positive control) which suppose to appear as single band ~700 bp on the X-ray film. Anyone having the same problem with me?Should I change to other brand of gel extraction kit?
I don't think you should change brands. A different kit would do exactly the same, as all a gel extraction column really does is bind to DNA.
The extra band is probably some kind of side product made at low level in the PCR reaction. Which is high lighted by the sensitivity of the southern blot. Heck it could even be your template. If your PCR product is concentrated enough, it can pool down contaminants.
My suggestion to get rid of the extra band is to re gel purify your insert. Run the 700bp product on a second gel, and give it a lot of running space. I think that should do the trick.