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Band diffusion of my PCR product in electrophoresis - (Jan/03/2006 )

Hi all,

I ran my PCR products, which are in the range of 100bp to 550bp on 1% TBE gel. What I got from the gel electrophoresis was a lump of "band" instead of one sharp band (like the one shown in the ladder or marker). The ladder looks good beside my sample lane though.
The lump of "band" shouldn't be primer dimer as it will start to show below the 300bp band.

Can anyone tell me what had happened to my PCR product?

Thank you very much in advance.

HaoHao

-haohao-

QUOTE (haohao @ Jan 3 2006, 04:25 PM)
Hi all,

I ran my PCR products, which are in the range of 100bp to 550bp on 1% TBE gel. What I got from the gel electrophoresis was a lump of "band" instead of one sharp band (like the one shown in the ladder or marker). The ladder looks good beside my sample lane though.
The lump of "band" shouldn't be primer dimer as it will start to show below the 300bp band.

Can anyone tell me what had happened to my PCR product?

Thank you very much in advance.

HaoHao



could it be DNA degradation?

-laurence-

when you mean a "lump" of a band, do you mean a smeary one?

if you have too many cycles in your PCR you tend to get saturation of amplification, ie: stackloads of DNA and could be a simple overloading of your PCR on the gel.

it could also be that your primer pair/PCR cycle conditions were not specific hence the wide band.

also the buffer your DNA was in upon loading would also affect the way it migrates due to the salt and ionic strengths. I have seen this doing restriction digests with methylation sensitive isochizchimers were bands of equal size seem to be of slightly different sizes, both digests were done in their respective buffers of course.

Nick

-methylnick-

Nick,

Yes, by lump, I mean smearing of my bands but not the type where one has a denatured sample. The bands are simply not sharp.

Can you exlpain more about "specific condition" for a primer pair/PCR reaction? What does it mean?

Thank you very much.

-haohao-