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
- - - - -

amp plates containing xgal/iptg with expression vector


  • Please log in to reply
6 replies to this topic

#1 biology_06er

biology_06er

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 55 posts
0
Neutral

Posted 03 October 2011 - 01:55 PM

Hi there,

I'm planning on ligating my gene into an expression vecot (Pet32a) and have to plate on amp plates--i made some up a while ago containing xgal/iptg so just wondering if i can use these...i know i wont get blue colonies..but having these substances added won't make any difference to the end result right?

#2 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 03 October 2011 - 10:10 PM

Hola, pET32a is an expression vector, so in plates with IPTG and with the correct construction, the induced expression could lead to lost of colonie because induction could kill your expression bacteria. Moreover, to use the white blue selection you need to have a lacZ activity, for that, cloning plasmids (as pGEM-T) and strains( as DH5alpha) have the two parts of the gene to get blue colonies when the beta gal is formed or white colonies when any insert avoid its formation.For all those aspects the correct method is made the cloning in a plasmid/host adequate, prepare some amount of plasmid to check constructions and when you have your cloning plasmid checked, subcloning in an expression vector, check the construction again and after transform an expression strain to have your whised protein. Buena suerte

#3 allynspear

allynspear

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
10
Good

Posted 04 October 2011 - 05:21 AM

protolder is sort-of right on this one, although it will be dependent on what cell type you are transforming into. The pET vector system is dependent on the presence of T7 RNA polymerase to drive expression of the cloned gene, and if you are cloning using a non-T7 expressing E. coli (Which you should be) like JM109 or DH5alpha, then even in the presence of IPTG, you shouldn't be inducing expression of your gene. All the IPTG does is remove the lac repressor from the lac operator, but without the T7 polymerase to make mRNA, you shouldn't get expression. That being said, there is always a chance that you can get non-specific mRNA transcripts synthesized, and the lac repressor being there helps keep background expression down. Since making plates is cheap and fast, I would say, just make some LB+Amp plates and call it a day, but if you are in a pinch, as long as you are NOT in BL21 or some other T7-expressing E. coli strain, you can plate on LB+Amp+IPTG+X-gal plates without too much risk.

Best of Luck.

#4 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 04 October 2011 - 09:54 PM

Hola, I apologize by my deep ignorance about your idea but I thought that I was right. Thanks for your explanation and have good luck with your experiment. The fact that I am a active forer doesn´t mean that I´m an expert, but I replied with the aim to help you.Thanks again

#5 biology_06er

biology_06er

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 55 posts
0
Neutral

Posted 04 October 2011 - 11:30 PM

Hey guys,

Thanks for both your explantations...allynspear: i read your post and was like ah ok, so i can continue using those plates..UNTIL I read your last sentace :P...BL21 is what I'm using haha...but it's all good..I just made a bunch of new plates with amp/chloramphenicol...didn't end up getting any colonies using those other plates anyway so thought may as well make a whole new bunch..

protolder: no need to be sorry at all :D..your answer was helpful as well :)

#6 allynspear

allynspear

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 83 posts
10
Good

Posted 05 October 2011 - 05:46 AM

protolder, I did not mean to offend, but I have accidentally done exactly what biology_06er almost did and had major problems, so I learned the hard way.

Biology_06er, I am wondering why you are transforming a ligation into BL21 cells, though. BL21 are an expression cell line that you normally would transform into once you have verifyied your clone. They don't usually give high quality mini/maxi prep DNA, and they tend to be more expensive than cloning-grade competent cells. It's not that you CAN'T do it, just wondering if there was some specific reason.

Best of Luck.

#7 biology_06er

biology_06er

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 55 posts
0
Neutral

Posted 05 October 2011 - 12:56 PM

Hi,

I first transformed into omnimax cells and then miniprepped (and now currently) trying to cut my gene out for my pbs vector and into an expression vector...it didn't work the first time (hence my other post regarding transforming straight into BL21 from pcr product)..I'm trying again today the gel purification techniquew as that is much better




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

©1999-2013 Protocol Online, All rights reserved.