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
- - - - -

size limit of plasmid origins


  • Please log in to reply
2 replies to this topic

#1 evolver

evolver

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 08 April 2009 - 11:09 PM

Hi all,

I am interested in prokaryotic (E. coli) origins of replication. Does anyone know of a reference giving the theoretical or empirical size limit of DNA that can be stably maintained from different origins of replication?

In particular, I am interested in the P15A origin. I have found a reference where it could support a 10.6kb plasmid. But, I would like to use this origin and a chloramphenicol resistance gene to maintain a 16kb piece of polycistronic DNA, final size of the plasmid would be about 19kb.

Can anyone say if this is in principle possible? or if I should go straight for a cosmid origin, like oriV which we know can support 40+kb?

Thanks a bundle!

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,437 posts
241
Excellent

Posted 09 April 2009 - 04:04 AM

This will probably work, but I don't have a reference. DNA of this size is more difficult to transform, and is fragile in solution. Avoid vigorous pipetting and vortexing. Why not move to a BAC plasmid origin?

#3 evolver

evolver

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 09 April 2009 - 08:13 AM

Why not move to a BAC plasmid origin?


I think a BAC origin is probably the best idea. But we don't have any in our collection at the moment (although I can probably get one from somewhere...), AND it just so happens that I have a plasmid with the P15A ori and Chloramphenicol resistance conveniently next to each other. So I can easily PCR it and incorporate some regions for recombination with the 16kb insert.

I think I will trust your and my gut feeling that this will probably work and give it a shot. It is less than a week to have an indication of success so not too much is lost in the case of failure...




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

©1999-2013 Protocol Online, All rights reserved.