I am trying to develop a flowchart to prepare my mammalian vector for transfection. This is my thought process so far:
- grow plasmid in a growth medium with a selectable marker (Ab resistant)
- extract and purify DNA
somewhere I am getting lost or confused and have the following questions
don't I need to confirm that the promoter, ORF, poly A and sites for ribosome assembly are present before I begin transfection? how would I do this?
do I need to cut the plasmid with a restriction digest and have it self-ligate?
thanks for your help