Weird Band gel migration - Molecular biology (Nov/22/2005 )

Hello, My problem is that after a PCR, I run in a gel for purifying the fragment of interest. I cut the band of interest and after I extract the DNA with a kit extraccion gel. Is very surprising what I see after extraction. I run the purified single band in a gel and 2 bands appear, one with the correct size and the other one smaller (the smaller one with quite intensity, almost as the correct one). My PCR product is very rich in A+T, the size is 1,6Kb, I think that could be secondary structure in the DNA, loops or thing like that. I think this structure doesn´t form in the PCR buffer reaction (NaCl, Mg) (in the first gel) but after extraccion the DNA is in water and form 2 structures. It is possible? Is any other reason?
Thanks a lot
It make me crazy

Just a thought, but have you checked that your gel purification reagents are free of contaminating DNA? I don't know if DNA contamination would give such an intense band, but it could explain why you cut one band out and get two after purification.

<have you checked that your gel purification reagents are free of contaminating DNA?>

it couldn´t be contamination because I am doing this with 4 different size PCR (but similar sequence homology AT rich) and all 4 after purification presents 2 bands, and the strange one is the different size. The most surprising thing is that the 4 PCR products are between 1,5 Kb and 1,7 Kb, and the distant between the correct band and the strange one is the same in all of them.
Look at the file

Thanks

hi,

what is your template for your PCR reaction. if it is RNA , it could be that you are seeing isoforms if any for your product of interest, since they are forming a basic pattern in all the four primers. just a thought.

check it out

How did you extract your band ?
Kiagen gel extraction kit

Did you try a restriction cut to see whether you hacve the correct profile ?
No, But I´m going to try it

what is your template for your PCR reaction?

The template is genomic DNA


Yes, I think it is just a migration problem and there is not mixed of inputs.

<< just try the restriction digest and will see>>

I did the restriction digest and it is correct. It is not any strange band. Pesji was right, It was only a migration problem.
Thanks a lot to everyone