posted by Restriction Enzyme Digestion .
I know it is a bit long and maybe difficult, please help me to answer these..
1) I have pET28a with insert inside. Insert is 400 bp in size. I want to check my insert with both single digestion and double digestion. I already isolated my plasmid but i do not know the next step. Please explain experimental set up for me in detail. How should I prepare my restriction digestion samples. How about agarose gel concentration and why that concentration? How will I compare my results, with what? And What am i gonna see on agarose gel? Is there anything that i should know about reagents or steps of the procedure? You know I have no idea about restrction enzyme digestion and you are my assistants, please explain me.
(you do not have to give specific names for restriction enzymes but you have to state all necessary assumptions. Ex: from where those enzymes cut your vector, especially for double digestion?)
2) Optimal time and temperature for digestion for each enzyme differs from each other. What are the criterias to determine optimal incubation temperature and time course for restriction enzymes?
3) I have empty pGEM-t cloning vector and i want to clone a gene inside this vector. Double digestion is necessary. Choose suitable restriction enzyme couple (it is up to you), find a web site to check whether those two can be used together and whether they are suitable with my vector. And find suitable buffer , duration and temperature for those two on web site. I want you to take step by step screenshot from web site and show the answers on pictures.