PEG 8000 precipitation/ gel electrophoresis - (Oct/19/2011 )

Hi!

So, I just run a gel with the plasmid sample I precipitated with PEG 8000 (washed the pellet with 70% EtOH, and resuspended in TE buffer), and it gives me this weird (high molecular weigh) structures on the gel.

This is the first time I've done PEG 8000 precipitation, so I don't know what's wrong?
Is this some residual PEG that didn't wash off?
Could that affect the ligation I have to do with this product?
Can I get rid of it by doing EtOH precipitation?

I get this pattern repeatedly on my gels. So it's not some artifact from the gel.

Many thanks,


photo:
Size of the left band is 10kb. This should be the size of the right band also, just that it has this weird smearing.

Looks like a genomic DNA smear to me.

It can't be. There was no genomic DNA involved.

It was a 10kb plasmid, cut with 2 restriction enzymes, PEG 8K precipitated, and washed with EtOH. Resuspended in TE.
I did the EtOH washing again. But the smear doesn't go away.

OK, thought you might have been doing a mini-prep.

I guess it is residual PEG then, or perhaps one of your RE's isn't working properly and you are getting some concatemers.

Yeah, I guess it's leftover PEG. I can't get rid of it even with multiple EtOH washing.
However, it also seems that it's not affecting my downstream ligation.
Which is good.
Thanks!

It looks to me like you may have pierced the gel with your tip and maybe injected the DNA further back into the gel so you are getting a weird pattern?

But I guess if your ligations are working, and you don't see it again, its just one of those things you can forget about!