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
- - - - -

Concepts behind plasmid prep


  • Please log in to reply
2 replies to this topic

#1 spellberg56

spellberg56

    member

  • Active Members
  • Pip
  • 13 posts
0
Neutral

Posted 24 March 2010 - 07:21 AM

Hello, I was curious to know why a plasmid prep isolates only the plasmid DNA and not the bacterial chromosome? Could someone explain how the different steps of a typical plasmid prep lead to the isolation of only plasmid DNA?

Thank you

#2 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,337 posts
82
Excellent

Posted 24 March 2010 - 08:34 AM

The chromosomal DNA is too much "wide spread" it can not rebind (or not in a correct why, it will "reconnect" but not good enough) , the plasmid DNA is not so big and can "reconnect" after denaturing.

Eum I know this sounds strange, a picture might explain it better.

The circles are plasmids, the "lines" are chromosomal DNA.

Posted Image

I hope you understand it?

So in short: you denature both chromomal DNA and plasmid DNA and then you renature it with the acid. And only the plasmid DNA can renature effectivly.

Edited by pito, 24 March 2010 - 08:37 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 klinmed

klinmed

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
1
Neutral

Posted 24 March 2010 - 09:21 AM

Hello, I was curious to know why a plasmid prep isolates only the plasmid DNA and not the bacterial chromosome? Could someone explain how the different steps of a typical plasmid prep lead to the isolation of only plasmid DNA?

Thank you


To put it another way:

With the most commonly used method (alkaline lysis) chromosomal and plasmid DNA are separated by differential denaturation/renaturing. Bacteria are lysed with ionic detergent (SDS) and NaOH. During lysis both types of DNA are denatured. Subsequent rapid neutralization (by addition of a concentrated potassium acetate buffer) allows only the covalently closed plasmid DNA to reanneal and stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed by an insoluble potassium-SDS complex. This is removed by centrifugation.

Further purification by anion-exchange (eg Qiagen columns) removes further contaminants such as salts and protein. This relies on selective binding/elution of the strongly negative charged DNA to a gel containing positively charged chemical groups.

Hope this helps.

Edited by klinmed, 24 March 2010 - 09:23 AM.





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

©1999-2013 Protocol Online, All rights reserved.