using piece agarose band directly on PCR - does it work? - (Apr/11/2008 )
perhaps this sounds strange but a colleague told me that you can prick a band on an agarose gel with a pipette tip or tooth pick, place it in a tube with PCR mix with nested PCR primers or the same primers used before and the fragment is amplified!
I haven't check it, but does anyone know if it works?
It would save a lot of work, time and money, right!
Yes, this certainly works. Don't take any of the agarose -- you just want a bit of the liquid from within the band stuck to the tip or the toothpick.
In general, I've had trouble with re-amplifying PCR products with the same primers. If possible, use nested or semi-nested primers if you need to amplify a product. The problem is that short products tend to amplify better than long ones, and even if you gel isolate, the short products predominate on a second amplification.
As Phage said, this technique works well. I have used a needle instead of tip or toothpick, to stab the gel band.
Mostly used for nested PCR but if your band-of-interest is lowly expressed and not the only product generated, you can pick the template and reamplify but as Phage said, this does not always work.
thanks for the answers.
I'll certainly try this. I always had to gel purify and then do nested PCR to get a really difficult product. By some unknown reason, with the first PCR I always get several unspecific bands, no matter how much I try to optimize it. this will, of course, simplify things.
just stab and amplify!