Gateway cloning-modifications to "entry" plasmid - (Mar/31/2014 )

Hi!  

I am planning a new set of experiments and would like to use the gateway cloning system.  At this point I only want to  test a few sequences for regulatory potential (but could see this going to a much more high-throughput screen).  I want to make this process efficient with as few steps as possible.  After reading up on the system, I am left wondering if you could engineer a final "destination" plasmid (your reporter construct) that contained the attp1 and attp2 sites flanking a positive and negative selectable marker.  Then simply clone PCR fragments flanked by the attB1 and attB2 sequences.  Essentially this would be the same as creating the entry vector, I believe.  Does this seem like it would work.  It almost seems too simple and I'm surprised that I haven't already seen this protocol somewhere and makes me think I am missing something.  Thoughts? Thanks!!!

I've never used it, and just read the wikipedia page on it...

Wouldn't creating the attb1/b2 sites just create an insert that can be inserted into the entry vector?  I think you would need to create the attP site for it to recombine with a destination vector.

yes, but I dont see the point of what you are doing.

In essence what you do is: generate your gene by PCR flanked with the attb sites , mix this with your "destination" (the donor vector, becoming an entry vector) vector (with the attp sites).

SO you would end up having an entry clone (your destination vector) with attL sites flanking your gene..

Is this what you mean?

Pito,

Yes that sounds like you understand my proposed plan.  I was just trying to make sure that this method would work because the system is set up to go through two vectors.  The overall purpose is to be able to PCR (with attb tailed primers) a bunch of different products and quickly clone each into my "destination" reporter vector (with attp sites) using the gateway recombination enzymes.  In the past I have done homologous recombination but that requires much larger homology arms with lower efficiency and is therefore less convenient.  Does that make sense?  Do you have a better alternative for this type of cloning?

lmh327 on Wed Apr 9 20:21:30 2014 said:

Pito,

 

Yes that sounds like you understand my proposed plan.  I was just trying to make sure that this method would work because the system is set up to go through two vectors.  The overall purpose is to be able to PCR (with attb tailed primers) a bunch of different products and quickly clone each into my "destination" reporter vector (with attp sites) using the gateway recombination enzymes.  In the past I have done homologous recombination but that requires much larger homology arms with lower efficiency and is therefore less convenient.  Does that make sense?  Do you have a better alternative for this type of cloning?

Eum, yes you can do this, but I am not sure why you would do it like this if you simple want to put your genes in a "destination" vector, there are more easier/cheaper/fasters ways to do this.

As far as I understand it you want to put some genes in a vector , right? thats all?

why not just ligate them into your vector? or use some other cloning system to put it in your vector?

with your proposal you end up with something like this:

vector - attL - your gene - attL - vector...

Now the attL sites are pretty long so you can not really use this system for a c terminal or N terminal linked system (for example GFP linked to your gene)/

Also: the promoter would be on your vector (I assume its not in your gene itself) so you will have vector - promoter (on vector) - attL - gene .... the attL would perhaps disturb this ...

I am not an expert on protein expression systems but it seems weird ...