Unspecific bands after gel purification, why? - (Apr/21/2005 )

Has anyone encounter with this: you get your PCR product as a bright desired band with some unspecific bands of bigger sized, then you cut out that desired band and do purification; but the purification product still contains multiple band/ light smear aboce the desired band. Why?
Something to do with 'too concentrated' DNA template in PCR?? (I don't think my cutting skill is too lousy... )

Has anyone encounter with this: you get your PCR product as a bright desired band with some unspecific bands of bigger sized, then you cut out that desired band and do purification; but the purification product still contains multiple band/ light smear aboce the desired band. Why?
Something to do with 'too concentrated' DNA template in PCR?? (I don't think my cutting skill is too lousy... )

Hi forest,

I have often seen this. I think, and may be wrong, but if you have a large band then some other DNA may get caught up with it and migrate larger or smaller than expected.

Scott

one professor explained this to me in a nice way. The bands that we see on an agarose gel are not distinct rectangular volumes of DNA. The bands are actually the peaks of a distribution of DNA (think Gaussian), where most of the DNA are present and where staining is most visible.

However, especially if bands migrate close to one another, cutting out the band for your desired fragment (the peak) may include the tail end of the Gaussian distribution of a higher or lower band.

The safest way around this to optimize your PCR reaction such that you eliminate these extraneous bands. The danger lies in that each extraneous band will have the same 5' and 3' end sequences (due to the primers) and if you incorporated RE sites on your primers than it is just as likely that these extraneous bands get cut and incorporated into your vector.

my 2 cents

hi
in case of you get rid of possibility of contaminants or degradation, i get the case of complexe stuctures. After a well done PCR, i saw three bands. Two of them disapeared when i heated my samples at 60° 10' prior to load an aliquot on agarose gel.

I think I'd found out the main reason for my problem. After I tried diluted template DNA (optimize by doing serial dilution), the smearing background and non-specific band in PCR product become less.
Thank all for contributing ideas =)

Problem again... I thought I'd gotten a single band finally, but in fact there are still two closely-placed bands. No matter how I optimize, my desired band always followed by another unspecific band, very close... what a headache.
Why...? My desired product is only ~600bp, and this is only a straight forward direct PCR for sequencing. Is my primer-pair so 'honoured' to match two regions in the genome? It is sad if I have to design/order another pair of it... I wish this is not true...
Help... =P

Hi Forest,

Two bands, esentially the same size from the one set of primers. Could it be possible that they are both what you're after but with a slight variation on splicing? I'd clone and sequence to determine what they are.

Cheers,

Scott