Effect of EtBr on plasmid supercoiling
Posted 28 March 2009 - 03:51 PM
I recently came upon this forum after searching the net for some answers to my dilemma. Unfortunately after hours of searching I haven't exactly found what I'm looking for and thus the reason why I need this forum's community's help.
When EtBr intercalates between the DNA it promotes an open conformation (relaxed). If this is compared to uncut DNA, how would you distinguish between the uncut supercoiled DNA and the relaxed form on an agarose gel (1%). In the uncut lane you will most probably observe separate bands for each of the different conformations (i.e. linear-circular-supercoiled), but what will be visualized when the EtBr intercalates between the successive basepairs? Will a ladder-effect be visualized (found several articles demonstrating this, only none of the authors used EtBr as an intercalating agent) as the SC band moves toward the relaxed band? I attached a photo of the gel I developed. It is of extremely low quality, I know...but unfortunately I still have to give a discussion of what I was theorectically supposed to see.
Any help in this regard will be most appreciated.
Posted 28 March 2009 - 11:41 PM
Posted 29 March 2009 - 06:42 PM
Posted 29 March 2009 - 07:08 PM
Thank you for the replies.
Posted 30 March 2009 - 12:17 AM
Sorry to be picky but why does the gel look so 'dirty'?
Edited by MaggieRoara, 30 March 2009 - 12:17 AM.
Posted 30 March 2009 - 12:58 AM
Clean the try in the same way.
Clean the lens with an appropriate cloth.
This will reduce the background specks you have.
Focus the lens to get a sharp image.
Load more in each lane. You can hardly make out the size marker.
Play around with the exposure