size of PCR product in PAGE - (Nov/27/2006 )

hi, this is a basic question. I was supposed to run a Gel shift Assay of 2 different gene promoters (with exact lengths) with a known protein repressor. But first, i tried running my PCR products into PAGE. However, i found out that they have different sizes. Does that mean that my PCR products are wrong, even though they appear as same sizes on Agarose gel?

Thanks for the replies

maybe because PAGE is more accurate than agarose now you can detect smallest differences.

somebody said to me that it might be due to AT tracts within the DNA that might caused the differences?how true is that?

Quite true. If you have runs of:

Poly(A)
Poly(T)
Poly(AT)

or

Poly(GC)

then you get significant bending angle in the DNA. Sometimes these can be more than a right angle over a reasonably short tract. This unusual secondary structure will cause changes inthe migration properties of the fragments.

However, this might not be what is happening here.

How long are your fragments? Are they short oligos or >100 bp?

Either way, if you create them by annealing then you can easily for secondary DNA structures such as hairpins, loops and others. These have a significant effect on the migration to such an extent that two similarly sized fragments can appear to be 100s of bp different in length.

I would suggest that you use much smaller fragment for EMSA. A 250 bp fragment will not give nice clean shifts. You can expect to see lots of smearing and aggregation.

Try something less than 50 bp and preferably about 25 bp. Just make sure these short probes contain the consensus binding sequence for your TF of interest.