How would one go about moving a gene from plasmid to plasmid - cloning (Jul/29/2005 )
I'm interning at UPMC Presby hospital. My PI wants me to move one gene from a plasmid in a bacteria to another plasmid. I am not sure how to do that.
Can anyone direct me to a protocol online somewhere which explains in detail how to cut the gene out of a plasmid (I know which restriction enzymes were used to put it together in the first place) and put it into a different plasmid? I also can isolate the plasmid as well as put a plasmid back into bacteria. I don't even know what this is actually called so it is hard to search for.
Can anyone direct me to a protocol online somewhere which explains in detail how to cut the gene out of a plasmid (I know which restriction enzymes were used to put it together in the first place) and put it into a different plasmid? I also can isolate the plasmid as well as put a plasmid back into bacteria. I don't even know what this is actually called so it is hard to search for.
New England Biolabs has a lot of good technical stuff about restriction enzymes at www.neb.com. And there is good info about molecular cloning (try searching for that term) at http://opbs.okstate.edu/~melcher/MG/MGW4/MG428.html
The Bible for this is Current Protocols in Molecular Biology ("the Red Book"). Don't know if it is online, but maybe your library or a colleague has it.
You're looking to subclone a gene. A google search of "subclone protocol .edu" (CLICK HERE) will give you tons of information.
Basically you'll cut your gene from one plasmid and run the digest out on an agarose gel to separate your gene from the rest of the plasmid. Since you know the size of the gene you're subcloning, you can excise it from that agarose gel, purify it, and ligate it into a new vector. Ideally, the second plasmid has restriction sites that are complementary to your gene (i.e. if you excised your gene from the first plasmid with EcoRI and XhoI then your second plasmid should have EcoRI and XhoI sites). If not, then it's a bit more complicated. Also, much of the cloning strategy depends on what you plan on doing with your gene. If you want to express protein, then you must make sure that your gene is still in the proper reading frame after subcloning it into the second plasmid.
Sorry, I got a bit carried away. In theory, that is how things work. I may have missed something because I'm in the process of my first real cloning experiment (still and undergrad), but those google hits will explain it all.
-Hank