Plasmid purification - (Feb/01/2006 )
I seem to have a problem with plasmid purification. I used Qiagen Megaprep columns to purify the plasmid and then I ran 1 microliter on an agarose gel to check for purity/intactness. Lo and behold, aside from the plasmid band, which I can see very clearly, there is a lower-size fragment (around 500 bp) as well. I spoke to a colleague who has worked with that plasmid before and apparently she once got the same result. When I did a restriction digest on the plasmid, I got the expected size fragments along with a faint 500 bp fragment.
Does anyone know what this could be attributed to? I thought that perhaps the plasmid is degrading but if that was the case, wouldn't I see many bands (sort of a ladder effect)? I'd appreciate anyone's input and help. Thanks
sure you're not looking at the supercoiled form?
agree with aimikins....
I have all 3 forms (supercoiled, linear and nicked circles) but higher up. I'm pretty sure that it's not the supercoiled form.
Could it be your plasmid without the insert, just self-closed?
The plasmid actually is not supposed to have an insert. It's a commercially-available plasmid, circular.
I will scan the gel picture shortly and attach it so you can see what I mean.