Gateway technology. - (Sep/29/2012 )
im new to molecular cloning. recently i came to know about the gateway technology.. i just was to know how far it is used in gram positive bacteria.
could anyone explain to me what is entry vector, donor vector, destination vector.s????
how will i come to know the compatibilty of plasmid to my bacteria???
more over what is the procedure to gene deletion from chromosomal dna using this technique????
hope somebody can explain in simple way???
The gateway technology is Invitrogen/Life Technologies' proprietary plasmid technology - you should check their website for information/manuals which will explain those terms better than I can. Those people that use it consistently say that it works well, but there are similar non-proprietary systems out there (e.g. bio-bricks) that may be better suited to your applications.
I havn't used either so I can't comment on the use in gram+ bacteria or their use for gene deletion.
me too heard a lot of its good efficiency. when it comes to R&D or PhD what is the main defect if proprietary technology is used???
munis on Sun Sep 30 10:56:04 2012 said:
main defect if proprietary technology is used???
Gateway looks simple, but as far as I looked at the principle, once shuttled, the gene cannot be shuttled again because the sites are lost (or more precisely there are some two steps, but that's it). Also I think someone on this forums was saying, that due to recombination technology, it can have some limitation.
So I rather used Origene shuttling system for my vectors. It's normal restriction-ligation system, but with specially selected RE on each side of ORF, that is unique for vast majority of sequences. But otherwise it's normal cloning. They have some commonly used destination vectors linearised and precut, ready to ligate, but unfortunately not the one I needed.
Problem is most of these systems are not compatible to each other, you buy ORF in one company, you can't use destination vector from other. So it's better to look at the vectors available, if they have what you need, because you can't change it.