VECTOR LIGATION WITH One RE - (Aug/06/2006 )
I want to digest my vector of 8 kb with one RE only so tell me the detail procedure how to purify the vector and dephosphorylated
it depends, really, on which RE you're planning to use, and how much plasmid you want to cut.
basically, you put a small amount of your RE into a moderate amount of your plasmid, with the buffer for that RE (and perhaps water, and/or BSA) Look up the catalogue or the product insert that comes with the RE. It will tell you specifically how much enzyme to use, and which buffers.
you then, usually, put this in a 37'C water bath for 2 hours.
to stop the reaction, the enzyme is usually heat inactivated. check with the product insert to see how much heat, and for how long. ie, 80'C for 10 minutes.
to phosphorylate, it depends if you want to use CIAP, shrimp, artic.... you have to be specific. each has their own protocol. I use CIAP. I put 1uL in my reaction, for 5 minutes at 37'C. and then gel purify.
other's use different purification methods. or heat inactivate it.
if you want more information, tell use which enzyme, and which phosphatase you want to use.
After RE digest, I hardly recommend to run your digestion in an agarosa gel, cut and purify your digested (lineal) vector. If you don’t do that, you’ll probably have a lot of background. Then, proceed to dephosphorylate…….
Please give more info on enzymes which u plan to use.
i usually dephosphorylate and then cut out of the gel. since CIP is not completly deactivated by heat. this can interfere with your ligation.