TOPO vs. Gateway cloning - Which is the better choice for large inserts. (Jun/07/2011 )

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

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.

phage434 on Tue Jun 7 19:54:15 2011 said:

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.)

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?

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.