Different conformation of DNA after gel extraction? - (Apr/06/2006 )

Hi everyone,

I was doing a gel extraction yesterday from digested plasmid. The DNA fragment that I excise from the gel is around 550bp and the other by-product of DNA fragments includes 2000bp and 1800bp. In other words 550bp is quite far away from the other bands on the gel.

However, after gel extraction of the 550bp fragment, I ran a gel to double-check the results and I got two bands, 1000bp and 550bp. I would like to know how is it possible that I can get that 1000bp band up there? Is it 500bp with other conformations? Any idea to fix that conformational change?

Thanks a lot in advance,

Haohao

Any chance the 550 bp fragment can ligate to itself? Was it a single enzyme, double site restriction? If so then you may have some amount of spontaneous ligation giving you a double-length fragment.

The 550bp DNa fragment has two different sticky ends as it was cut by two different enzymes.

Thanks

If we assume that there could be some spontaneous ligation (is it true? can it happen?), then 2 plasmids could ligate, even if there are two different enzymes involved. You just have twice less opportunities? No?

Hi.
Could probably be that 550bp fragments with "sticky end A" forming complement hydrogen bonding with "sticky end A" of the other 550 bp fragment, though it has another "sticky end denoted by B". This could happen if DNA concentration is high enough to favour such interaction.

You could try, after digestion or in your case after gel purification, by heating briefly (1 min) at 65 Celcius and quickly place on ice, 2-5 min before adding 6X loading buffer and running in agarose gel. If the 1 kb band still persist, it's a mystery yet unsolved.

Cheers

I will give it a try and get back to you guys.

Thanks again, really appreciate it.