PEG 8000 precipitation/ gel electrophoresis
Posted 19 October 2011 - 06:39 AM
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.
Size of the left band is 10kb. This should be the size of the right band also, just that it has this weird smearing.
Posted 19 October 2011 - 11:40 PM
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.
Posted 20 October 2011 - 02:49 PM
I guess it is residual PEG then, or perhaps one of your RE's isn't working properly and you are getting some concatemers.
Posted 23 October 2011 - 01:28 PM
However, it also seems that it's not affecting my downstream ligation.
Which is good.
Posted 23 October 2011 - 07:38 PM
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!