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
- - - - -

Restriction endonuclease


  • Please log in to reply
4 replies to this topic

#1 aztecan princess

aztecan princess

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 01 June 2009 - 12:06 PM

Do Restriction endonuclease like EcoRI cut only double stand DNA? Or they cut single stand DNA too???

Thanks!

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,420 posts
238
Excellent

Posted 01 June 2009 - 02:10 PM

Double stranded only, and not too close to the ends.

#3 aztecan princess

aztecan princess

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 01 June 2009 - 02:28 PM

ok, thanks, I just wanted to be sure. :P

We are doing RT-PCR after plasmid tranfection, RNA extraction and DNasa treatment. The problem is that we are not able to eliminate all the plasmid and we have amplification using only RNA. So I think We could digest with a restriction enzyme cutting into the gen before RT-PCR to avoid false positive amplification. What do you think??

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,420 posts
238
Excellent

Posted 02 June 2009 - 08:01 AM

DNAse would be the normal choice. With your strategy you would need to worry about the RE staying around in your RT reaction, and cutting the final dsDNA product after amplification. If you can heat kill the RE, then this might be an OK strategy. I'd try the DNAse first.

#5 aztecan princess

aztecan princess

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 02 June 2009 - 01:50 PM

DNAse would be the normal choice. With your strategy you would need to worry about the RE staying around in your RT reaction, and cutting the final dsDNA product after amplification. If you can heat kill the RE, then this might be an OK strategy. I'd try the DNAse first.


Actually, what we are doing is trying with DNasa, then digesting with the RE, and then heat inactivation before RT.
The problem, as I told you is that using the DNasa treatment is not enough, as we canít eliminate all the plasmid. We tried two different enzymes and double digestion time with no success.




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

©1999-2013 Protocol Online, All rights reserved.