Cloning into GATEWAY system - (Oct/03/2007 )

Hello, everyone!
I'm new here and into cloning world...
Can you help me GATEWAY cloning?
I'm very interested into problems with this approach. Can you tell me everything (the dark side of GATEWAY?)?
Thanks a lot!

(Sorry for the bad english)

Mel
(trying to get 15 clones until oct15 !!!)

I've made a number destination vectors using their vector-conversion kit. Most of the issues I've had are fairly simple technical problems that you can troubleshoot your way through. The constructs I've made w/ gateway cloning work perfectly and the process was faster than conventional cloning, especially considering the larger plasmids our lab uses.

Can't really help you without some more specific questions, but some of the "darkside" in my experience w/ Gateway:

- less than 100% effeciency, ie after the LR reaction not all my colonies are the right construct

- the competent cells used to propigate the destination vectors and empty entry vectors are not robust and need to be babied (ie grow for 24 hours to get colonies, need to grow in SOC for an hour which we don't need to always do for normal competent cells)

- Low DNA yields from said cells, however we make large vectors (~15kb after putting in the gateway cassette) so that may have something to do with it. In any event, this can be easily dealt with by simply following the low-copy # protocols for your midi/maxi prep.

- the empty entry vectors have actually mutated when I grow up more to make a stock supply, basically the ccdB region in the MCS doubled in size. I was able to avoid this by transforming the cells w/o the SOC grow-up, only got a few colonies but they were the right plasmid!

- not really a dark side, but make sure you maintain chloramphenicol selection when propogating your destination vector, otherwise the cells tend to kick out the plasmid and you get terrible dna yields.

Its also worth noting that I encountered alot of these during my first year of grad school, so some of it may have just been some bad karma.

As I said, these are little things that you can troubleshoot, the flexibility and speed of gateway cloning makes up for it I think. Good luck!

Thank you very much for the answer!
We are new in gateway technology and when I was reading the manual I was wondering... can anything be so good as the gateway? That´s why I think about a "dark side" not written at the manual... but it seems to work well, doesn´t it?
I´ll soon try to clone 15 genes in order to resolve the protein structure...
Thank you again!
Mel

Thank you very much for the answer!
We are new in gateway technology and when I was reading the manual I was wondering... can anything be so good as the gateway? That´s why I think about a "dark side" not written at the manual... but it seems to work well, doesn´t it?
I´ll soon try to clone 15 genes in order to resolve the protein structure...
Thank you again!
Mel