the problem in gel purification - (Oct/13/2007 )
i am being obsessed by the gel purification now.
i am doing the PCR ,after it finishing ,i run the gel to investigate the size of the product,
and position of the band compared the marker is what i want
so i do the gel purification ,after that,i want to check the efficiency of my purification ,so
i run another gel
and the strange thing is that the band seemd become larger than it had been ,
and i have repeated it for several times ,the band of the purificated products are the same
but they are all larger than the products before purificated ,so
where the problem lies ,i want to hear your analysis.
without the purificated product ,i can't do further research,
Are you not getting very good PCR? I mean if you are getting a smear type, then may be you are cutting the nonspecific band. try to resolve completely your band of interest, then cut it carefully and purify it.
What is the size of your product and what method do you use to purify your band? I agree with mysterious, that you should well separate your band and then cut the band if you have not done so.
can you show the gel pic of both the gels? It is better to run a gel further before cutting out the PCR bands.
You can test this by keeping a small amount of your original PCR reaction and running it along with the isolated fragment on the second gel. Salt concentration and DNA concentration can easily affect the running speed of a band, so it is not too surprising to see results such as this. If the concentrations are similar, then there is something peculiar going on, but I doubt there is a problem.