Gateway cloning - help (Feb/22/2006 )
Hi guys,
very nice forum indeed.I am new to the field of molecular biology. I've just joined a lab and finding it very difficult. Not really difficult but don't know why I am not getting the results in PCR, Miniprep etc. when everything is standardized? Well I am only trying my level best or else what can I do? My guide has given me a topic of Gateway cloning to prepare. Actually he wants to buy the product. I read about this but I just wanted to ask is anybody there using it? How is it? I mean what are the advantages or disadvantages? I'll be very thankful to u guys please help me.
Thanks
i started working with it not long ago, so i can not provide to much experiences. up to now it really works fine for me. of course you have to keep in mind, that you might be forced to make new vectors for your needs, which can be a lot of work. but once you have your entry clone you can go anywhere. also the negative selection with the toxic gen works fine in my hands. you should think a lot before starting the gateway system, especially which e.coli strains to use and which antibiotics you might need (the DONR and DEST vectors carry different resistance/more than one resistance). also keep in mind that depending on your destination vector, you might have so additional aminoacids on your protein.
more general remarks:
- check the date of the manual, if you download one. there are at least two available, be sure to get the newest one.
- buy clonase II (which comes with its buffer already)
- BP clonase should be stored at -80°C!!! on the box -20°C is stated (I reported that to them, maybe they changed it...although I doubt that)
- LR clonase should be stored at -20°C
) I'll make some general introduction to recombinational cloning. I think within several years it will be a very often used standard procedure, my be more common than classical cloning.so, general advice to everyone, who has no idea about recombinational cloning, read:
"Many Paths to Many Clones: A Comparative Lookat High-Throughput Cloning Methods" from Gerald Marsischky and Joshua LaBaer, Genome Reasearch 2004
Cheers,
Kersten
Hi Kersten 
Thanks for giving me such a great info. Well I'll keep all the points in my mind. But I don't understand the funda behind this actually. As I told before I am new to the practicals although I've read all the things in theory. But......
I just understand first we go for the cloning of a particular gene that too in fragments, then we ligate all the fragments, and then we go for the expression. I read about the Gateway cloning and also got that there are many advantages of this technology but not really understood. Can anybody please explain me with the comparison of the normal cloning. I mean how some steps are reduced in this?
Please someone help me.
okay ... as I said, it might be worth writing from a more general point of view ... but times short man
so, in "classical" cloning you have to find restriction enzymes that do not cut in your gen and that cut your plasmid only once. than you pcr, you digest, you ligate and screen for positive clones. especially the last step can be quite a pain in the ass. If everything works out fine, and you have your clone, nice... but now you want to have your gen in a different plasmid (subcloning) ... so everything starts from the begining. 
now when using gateway cloning, you use recombination to transfer genetic information between vectors. no restriction/ligation. also recombination exchanges the information that is in the plasmid (between the recombination sites) with the genetic information that enters the plasmid. in gateway cloning the information that is already in the plasmid (and will be reomoved by recombination) encodes for a toxic gen. So, if there are plasmids where on recombination takes place, cells that'll have this plasmid will die. so in theoryyou end up with something like 99% efficiency for recombination. in my hands, i never recieved an "empty" clone up to now.
also, when you have your entryclone you can go from that to any other plasmid, that contains recombination sites, without laborious subcloning. 
so basically go anywhere ... e.coli, yeast, mice, in vitro expression ... you name it. of course as long as a compatiable plasmid is available. but if not you can prepare them quite easily (by "classical cloning" that is).
the cloning with severeal fragments you refer to (if i understand correctly) is another advantage. you can have different recombination sites, and by that have different fragments from pcr which will go into one plasmid by one step recombination and also they will be in one order only. but for cloning of one gene only you don't need this.
i hope, this is not all to confusing, because i just write it don't without to much thinking 
if there are more question, feel free to ask.
Hey Kersten
Thanks very much. Its really good that u spend some time for newcomers like me. I got the basic idea. If u know some good sites about this that also i'd like to know.
thanks
google might help a lot, also check the article above. besides that:
http://www.alab.com.pl/gateway/gateway_dodatek.ppt
http://www.invitrogen.com/Content/Online%2...atewayhome.html
Hi Kersten
Thanks again.The first link is not working on my system, I don't know why? 
.Anyways second link is very good. I've a confusion, Why do we add Proteinase K in both the reaction? its given that it helps to improve the efficiency of transformation, but how?
Thanks
the first link points to a powerpoint presentation... do you have powerpoint installed?
the proteinase K destroys BP/LR clonase. I guess that's important to not have any unwanted recombinations.
ya we do have powerpoint installed in our lab, but don't know wats the problem?
ok I wanted to ask one more thing and that is , do we have to construct the vectors in this gateway cloning or they are available in the kit?
Thanks
plz check the invitrogen homepage. there are a lot of possible destination vectors avaiable -> check here for example. of course, if they do not suit you, you can design your own ones.