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
- - - - -

TOPO vs. Gateway cloning


  • Please log in to reply
4 replies to this topic

Poll: TOPO vs. Gateway cloning (1 member(s) have cast votes)

Which would you choose to get better efficiency

  1. TOPO-TA (1 votes [100.00%])

    Percentage of vote: 100.00%

  2. Gateway technology (0 votes [0.00%])

    Percentage of vote: 0.00%

Vote Guests cannot vote

#1 nimrod337

nimrod337

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 07 June 2011 - 08:57 AM

Ok, I have been at this for a while now, and I think it's time to upgrade my technology so I can get some results.

I am attempting to clone a gene of interest from potato. The fragment is about 6.5 Kb, and it includes the coding sequence, and 3Kb upstream (presumably the native promoter.)
The overall goal is to get this chunk of DNA into a binary vector and transform plants with it.
What I've already tried:
-a-tailing and cloning into T-Easy (3Kb vector.)
- adding RE sites on my primers, and digesting the PCR product, and the vector (9Kb), then ligating using T4. (yes I added 3 extra bases at the front of the RE sites so that the enzymes could digest properly. (almost every step was checked by running on a gel, and extracting the desired bits.)) (REs: SmaI, and SpeI.)

So my PI says it might be time to bite the bullet, and upgrade our tech to try and get this guy working. So I am looking into TOPO cloning, or Gateway cloning as a more efficient method. What do you think would work the best? It's a large insert, and a semi-large vector. Thanks for your input.

-A

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,507 posts
254
Excellent

Posted 07 June 2011 - 11:54 AM

Given that you are choosing a restriction enzyme, why would you choose SmaI, which leaves a blunt end? A cohesive end ligation would make your life much easier.

#3 nimrod337

nimrod337

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 07 June 2011 - 01:21 PM

Given that you are choosing a restriction enzyme, why would you choose SmaI, which leaves a blunt end? A cohesive end ligation would make your life much easier.



Indeed. That would make my life easier. Or if both enzymes worked at the same temperature. However, that is the nature of the beast. I did not choose SmaI, it just happened to be one of the only REs that was not found in my insert or vector when I started these shenanigans. (correction: it was one of the only enzymes that was found only once in the vector, at the MCS. And not at all in my insert.)

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,507 posts
254
Excellent

Posted 07 June 2011 - 03:48 PM

I'd do a non-directional SpeI cloning and analyze to find the half of the clones in the correct orientation (if I cared). You can change the MCS by doing PCR with whatever sites you need on your vector, so you shouldn't e limited by MCS enzyme sites. What's the next step you need to do?

#5 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 09 June 2011 - 07:08 PM

I would suggest that SmaI be substituted with XmaI.
Both XmaI and SmaI cut the same restriction site. However XmaI cuts at 37 C and produces an overhang.

I personally would prefer the DNA ligation. You are not limited by the MCS on the vector plasmid. You can PCR amplify the vector. And you can then add any restriction site you so desire on the primers. I would also use 6bp overhang rather than 3bp.

I believe the Gateway system is more efficient... However I have found that it is rather difficult to further manipulate a DNA fragment once it has gone into a gateway vector. There are rather few restriction sites in a gateway vector.

thus I think that TOPO cloning would better serve your purpose.
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.