Hello, everyone. I'm doing a study in genetics at my school, and I've run into a bit of a roadblock.

 

Part of my study involves performing subcloning. I understand that a gene of interest must be isolated from a parent vector before it can be ligated into a new vector, and that the ends of the gene of interest and the vector you're trying to insert it into have to have been cut by the same restriction enzymes.

 

For example, if you have plasmid A and plasmid B, and you want to take a gene from plasmid A and insert it into plasmid B, you have to do a restriction digest to cut the gene away from plasmid A, as well as a restriction digest -- using the same plasmids as you used with plasmid A -- to open plasmid B such that the gene can then be inserted.

 

 

So I'm wondering: What do you do when subcloning if your gene of interest and your vector don't have any complementary restriction sites?

 

Thank you.

 

Solved: PCR using primers with restriction sites built-in, or, alternatively, blunt-end cloning.

 

Edited April 16, 201213 yr by Hawk Pidgeon

Precisely. Also you can just use restriction enzymes and then blunt the ends for blunt end cloning (hence omitting PCR). Also there are vectors that allow T-A ligation (as Taqs usually introduce a A overhang).