No DNA after gel extraction - (Jul/22/2013 )

I am facing problem in gel extraction of my insert of 1.5 kb. I load about 15-20 microliters of PCR product in the well, there is always a very sharp, dense band, but after gel extraction there is nothing or very little DNA. I have used different kits and followed the protocols of the manufacturers, but cannot get significant amount of DNA. Can any expert identify my mistake and suggest me some tips?

awan on Mon Jul 22 13:07:07 2013 said:

You should also ask why you are doing this. If you have a single sharp band, then gel extraction is just a bad idea.

Since you are getting sharp band then you can go directly for the PCR extraction. Just add the solution into the PCR product and extract.

there is a faint band at about 6 kb also. I have been doing direct PCR clean-up previously. but now I want to get a more purified DNA for cloning...I just followed the kit suppliers, Last one was by Qiagen.

how are you determining where to cut? are you cutting while viewing the band on a uv illuminator?

you may be destroying the dna with the uv or you may be missing the band (or most of it) while cutting.

also to consider, a very sharp band may not contain a lot of dna.

yes, I view on UV illuminator for a few seconds, just draw lines by scalpel, and then cut the band on my desk.
I load 20 microliters per well, is it more than a column capacity? is there any chance of column choking?

the protocols that come with the kits should tell you the loading limits of the columns.

or, are you referring to the gel lanes? then there shouldn't be a problem with your load. if there was, you would be able to see it when you looked at the gel.

Thanks dear experts. It was good experience discussing the problem with you people. I shall keep in mind these points next time.

Is there any way of first optimising the PCR (Tm and/or Mg2+ conc.) so that the 6kb does not appear, then with the 1.5kb only you can easily do QIAGEN PCR clean up kit?