I plan to clone a bacterial gene. I ordered the plasmid vector pGEX but I just learned that the order will arrive in six months. Who can offer me a protocol so that I can build a cloning vector myself to save time? All I know is that a vector must have a promoter, specific sites for restriction enzymes, and an antibiotic sequence that would allow me to make a selection.
Building a vector from scratch is do-able, but it's a tall order. It might be a pretty fun learning experience, but there's a lot of potential for failure... It's going to take a lot of testing, and in the end be pretty expensive. For 60 dollars you should be able to find a bacterial expression vector on addgene.com
Building a vector is actually quite simple, if you have the parts on hand. For a simple cloning vector, all you need is a proper replicon, e.g., ColE1, a gene conferring resistance against an antibiotic of choice (I would suggest ampicillin or kanamycin) with a proper terminator, and a few unique restriction sites, one of which should be for a blunt end cutter.
Of course, there are many extensions possible for this simple design: a promoter and RBS for an expression vector or a lacZalpha gene with promoter to allow for blue/white selection. But for all this, you need the proper parts. So I agree with Ed: either find a different plasmid on addgene or ask around. Many colleagues will share strains and plasmids upon request.
Thanks. It's better to change the vector as i can see handling is a bit tricky
pGEX is a very common vector. I would find a publication using this vector and email the corresponding author. You may find one with your desired gene already cloned in, otherwise, I would request any other cloned gene as long as it was clear how they had inserted their gene so that I could easily excise that gene and replace with my gene instead.
Dear Meirem,
if you have in your lab any other vector (even with insert inside), take the miniprep- digest the insert out- make transformation- collect white colony (empty)- make miniprep and you have a vector without ordering anything
and if you don't have any vector in your lab- ask from any other lab 1ul of vector and make mini/midiprep to have nice amount
good luck
I think I'll work with pbluescript vector it's better. Thank you for your reply
Vector NTi is one of easiest ways to design clones. I think that there is a student version free (but I'm not sure).
Most important think is to know the poli-linker restriction enzymes of the pGEX and check that these enzymes are not present in the gene. If you have different options, I would choose two different enzymes (to avoid religation) and if it is possible with compatible buffers. After that, you should desing your primers including the restriction choosen.
why bother...Addgene has thousands of vectors and they can arrive in days depending on where you are. Call Addgene or email [email protected] to get advice for your purposes.
http://www.addgene.org/empty_backbones/
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