Supercoil migrates slower than linear plasmid? - (Sep/24/2006 )
I purified my plasmid using sigma miniprep kit.
Then I digested the plasmid.
I run a gel with both the plasmid and the digested plasmid.
On the gel, there are three bands in the lane for my plasmid.
One weak band, migrated fastest. (should be linear)
The other weak band, migrated slowest. (nick)
A very strong band, migrated between these two weak bands. (should be supercoil. It is close to the nick one.)
There is only one band in the lane for digested plasmid.
The size of this band is the same as the fastest weak band in the lane for my plasmid.
And the size is correct.
Have you ever meet this problem?
It seemed that supercoil migrated slower than the linear.
I wonder what is the reson.
I added 4ul EB(1mg/ml) in 30ml gel and 2ul EB in 200ml running buffer.
The voltage I used was 110V.
The running buffer is TBE.
Hope you could help me.
Thank you very much.
how do u know that the fastest band is linear in the uncut plasmid !
what concentration of gel u used?
You're assuming you've correctly identified the bands you're seeing in the uncut lane...
There are several additional forms of plasmid DNA then the three most people mention. Plasmids can exist in dimeric or other multimeric forms as concatamers or catenates, circular with different degrees of coiling, and linear, and can also possibly include denatured forms of all of these (the denatured forms occur due to remaining too long in alkali conditions during lysis, and are resistant to digestion). Additional bands may arise due to contamination with bacterial chromosomal DNA (a consequence of being too vigorous during lysis or neutralization steps).