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 a DNA prep on DE3


  • Please log in to reply
2 replies to this topic

#1 Dr_Watson

Dr_Watson

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 10 November 2010 - 01:06 PM

Hi all,

Here is my problem, I transform a plasmid into a cell containing the DE3 lysogen. The DE3 allows the expression of the T7 RNA polymerase after induction with IPTG. So I've transformed several plasmid into the cell and with one specific plasmid I don't see any expression of the T7 RNA polymerase after induction with IPTG.

I always have a control to check that the problem is not the IPTG or the Ab during the W.Blot. With different plasmids the expression is always of the same intensity. And that's what I don't understand as the type of plasmid shouldn't interfere with the DE3, should it?

So my question is: can the DNA preparation be the cause? I mean, if the preparation is not that pure (even though it's from a minikit) can it be a problem? (like using a too large volume of cells for the miniprep...)

thanks for any kind of help, suggestion, positive vibes...
Dr_W

#2 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 10 November 2010 - 08:28 PM

might there be a mutation in that particular plasmid
May your PCR products be long, your protocols short and your boss on holiday

#3 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 10 November 2010 - 10:31 PM

Hola, I understand that you see your recombinant protein band before induction and adding IPTG the expression donīt increase. This is because there isnīt any repression of expression, levels of cAMP are high and the promoter/operator is open. Why do you try to repress the expression with glucose 0.1-0.2 g/l or better use a (DE3)laqIq strain? Good luck




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

©1999-2013 Protocol Online, All rights reserved.