Originally Posted by arunab129
can any one help me in my problem. i am working with pET DUET SUMO vector to clone my gene. and my restriction sites are BamH1 and Kpn1.i cant able to get the clone. i have a doubt whether double digestion of vector is occuring with the two restriction enzymes or not. can any one please tell me how can i perform double digestion.
The most important thing you have to check are the enzymes activity conditions. Not all the digestion enzymes can work on the same buffer, or at the same temperature.
Some New England Biolabs digestion enzymes (the ones I usually use) can be used together within the same temperature, time and buffer, so you can simply add both enzymes to the NEB Buffer, add the BSA, DNA and then incubate the reaction and have a double digest in only one reaction.
If you want to double digest with two enzymes that don't act at the same conditions, or have star activity or something that makes your one-step double digestion troublesome, you will have to digest with one enzyme first, then purify the DNA and digest with another enzyme, then re-purify to ligate with your vector to transform.
But, are you completely sure the double digestion step is the one compromising your cloning? There are many other steps than can affect your cloning, like the ligation step, the transformation step, the efficiency of your bacteria, etcétera.