Hi everyone, I am back for help again.
I have basic cloning question and I would like to ask detailed explanation for the future reference.
I have ORF cDNA clone of my gene of interest in the cloning vector (pGEM-T vector). Of course I want to transfer it to make expression plasmid (possibly pEF-BOS).
I guess this should be very simple. In the instructions, manufacturer says the coding sequence can be easily obtained by digesting the vector with proper enzymes and it also can be amplified by PCR.
What I understand is that I can choose an enzyme and cut the vector and the empty plasmid I want, than I can do ligation and transformation in E.coli.
My question is...which enzyme to choose? Can someone explain to me in detail what is important to consider when choosing enzymes (I guess this is very basic so I hope I am not taking too much of your time)? And I will have to do PCR also right?
Thank so much!