Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Effect of EtBr on plasmid supercoiling


  • Please log in to reply
5 replies to this topic

#1 BigB

BigB

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 28 March 2009 - 03:51 PM

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 Thumbnails

  • DPCF0152.jpg


#2 Sciurus

Sciurus

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 28 March 2009 - 11:41 PM

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.

#3 MaggieRoara

MaggieRoara

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 29 March 2009 - 06:42 PM

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

#4 BigB

BigB

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 29 March 2009 - 07:08 PM

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 [EtBr]; 3. 0.5 ng/Ál [EtBr]; 4. 200 ng/Ál [EtBr]; 5. 500 ng/Ál [EtBr]; 6. 750 ng/Ál [EtBr]; 7. 1000 ng/Ál [EtBr].

Thank you for the replies.

#5 MaggieRoara

MaggieRoara

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 30 March 2009 - 12:17 AM

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

Edited by MaggieRoara, 30 March 2009 - 12:17 AM.


#6 molgen

molgen

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 167 posts
0
Neutral

Posted 30 March 2009 - 12:58 AM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.