plasmid preparation - (Nov/20/2008 )
QUOTE (AS mikkel @ Nov 21 2008, 04:35 PM)
I did an electrophoresis of the 50X dilution of my plasmids and it looks much nicer. But the size is still not the same as the expected ones. I guess it is due to the sepcial structure of the plasmids, circle or super-coil form will both effect the migration rate and make them different from the one of linear DNA. Is that the right explanation?
Yes, that's why I said you should digest with an enzyme with a unique site. You will linearise the DNA, if it's plasmid.
Again, if you had genomic DNA, there is absolutely no reason that it would all shear to the same size; therefore you have plasmid. It's bigger than you were expecting, but it's plasmid. The genomic DNA gets precipitated during the prep. You should find more details in the pack insert of your kit.
Some simple questions:
What is the expected size of the plasmid? What are the markers you're using? How big is the insert? What percent gel did you run?
By the way, rockofbio, why do you say there are two layers of agarose? If you're worried about the two different background shades, that will be because the EtBr has migrated out of most of the gel (remember it runs in the opposite direction to the DNA).
-swanny-