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
- - - - -

Cloning a whole gene in pBAD (any comments pls)


  • Please log in to reply
4 replies to this topic

#1 arvinsign

arvinsign

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 09 August 2009 - 06:18 PM

Hi..i would just like to ask for comments. Im planning to clone a whole gene in a pBAD plasmid. In this case i dont want to use the pBAD promoter, but instead the native promoter of my gene, so in short its a promoter + ORF. Im having problems with finding good restriction enzymes sites in other plasmids and i i like the MCS of the pBAD. So i want to clone a whole gene downstream of the pBAD promoter (hoping that the presence of the pBAD promoter upstream will not in any way affect the expression of my gene). Do you think this will affect the expression of my gene? Thanks

The upstream sequence of my gene will be around 250 bp from the native +1 site.

#2 fishdoc

fishdoc

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 272 posts
2
Neutral

Posted 09 August 2009 - 07:31 PM

Hi..i would just like to ask for comments. Im planning to clone a whole gene in a pBAD plasmid. In this case i dont want to use the pBAD promoter, but instead the native promoter of my gene, so in short its a promoter + ORF. Im having problems with finding good restriction enzymes sites in other plasmids and i i like the MCS of the pBAD. So i want to clone a whole gene downstream of the pBAD promoter (hoping that the presence of the pBAD promoter upstream will not in any way affect the expression of my gene). Do you think this will affect the expression of my gene? Thanks

The upstream sequence of my gene will be around 250 bp from the native +1 site.




Insert your gene in the opposite direction as you would if you were cloning off the pBAD promoter. Alternatively, you could put a transcriptional termination sequence upstream of your promoter so that the pBAD promoter doesn't read through to your gene.

#3 arvinsign

arvinsign

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 10 August 2009 - 07:52 AM

thanks fishdoc. i'll insert it in the opposite direction. I'll let you know the results later

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,504 posts
252
Excellent

Posted 11 August 2009 - 03:57 AM

You should not be limited in your thinking about the sites available in a particular MCS. It is very easy to design primers that will amplify a vector containing any restriction sites you'd like to see. Just remember to put sufficient 5' overhangs to eliminate cutting problems, then treat the vector PCR just as you would an insert PCR reaction.

#5 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 11 August 2009 - 05:35 AM

as an addition to phage434 suggestion. As you can PCR amplify the vector to add any restriction site that you desire, you can also eliminate the pBAD promoter that you do not want to use. That would eliminate the double promoter problem.
May your PCR products be long, your protocols short and your boss on holiday




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

©1999-2013 Protocol Online, All rights reserved.