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Effect of EtBr on plasmid supercoiling - (Mar/28/2009 )

Hi!

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.

Many thanks,

Barend.
Attached Image

-BigB-

As far as I understand EtBr doesn't significantly uncoil plasmids in sc formation. However, staining will be weaker in sc than in open conformation because the sc form prevents intercalation of EtBr.

-Sciurus-

Could you perhaps label the lanes on your gel. It is also rather confusing what the aim of your experiment is....

-MaggieRoara-

The aim is to demonstrate the effect of EtBr on the supercoiling of plasmid DNA. The lanes from right are: 1. Lamda DNA/ EcoRI + Hind III marker; 2. 0.2 ng/Ál ; 3. 0.5 ng/Ál ; 4. 200 ng/Ál ; 5. 500 ng/Ál ; 6. 750 ng/Ál ; 7. 1000 ng/Ál .

Thank you for the replies.

-BigB-

Supercoiling is probably there in your sample, but you might not be able to see it. Try repeating the experiment with more DNA. Supercoiled DNA is also "lighter"(as in brightness) than 'usual' DNA. So you need to increase your DNA amounts. Load a 1kb ladder, 100bp ladder and a linearised form of your plasmid (use only one RE) and run till your linear form of plasmid is near the end of the gel. Supercoiled plasmids are much much slower in migration, so it might take some time before you see the DNA. It is important to load more DNA so that we can see the small percentage of it that is supercoiled. I trust that the DNA samples were not digested before being loaded other than for lane 1.

Sorry to be picky but why does the gel look so 'dirty'?

Good luck! :P

-MaggieRoara-

Clean the surface of the transilluminator with distilled water.
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

-molgen-