extraction of DNA from agarose gels - (Jun/12/2006 )
I am using agarose gels to analyze my PCR products. I need to extract the desired products from agarose gels. To me, it is hard to locate the bands with UV light and cut them (harder than dealing with polyacrylamide gel). I just had a failure of extraction, which I guess is because I made a wrong cut. Can anyone give me some tips? Any advice would be really appreciated.
I'm guessing you've got a gel extraction protocol/kit, and your PCR is working reasonably well. Why do you need to extract from gel? Do you have a dirty product?
You can use EtBr and UV to see your band, just do it reasonably quickly, and be sure to use long wavelength UV, even though the intensity is reduced. Once the band is cut out, your can use the kit, or if you don't have one, chop the slice into little pieces, freeze it and spin down in a microfuge, if you have tubes with a fiilter unit (we always used Z-Spin tubes, can't recall supplier). Then just EtOH and ammonium acetate precipitate.
What's wrong with using polyacrylamide gel to see your band? Again, you can use EtBr and UV.
To locate the DNA bond, you can also use Gelatar, which can stain the DNA bonds more conveniently than EtBr. Gelstar(the product name can be purchased) can stain short ss-DNA as well as long ds-DNA. If you still worry that the dye will contaminate your sample, you can use HPLC for purification.
just checking that you are cutting with the UV still on rather than estimating the location of the bands? (wearing a UV face guard etc....)
(sorry if this is obvious, not trying to be patronising)
this has been discussed in a previous thread