Advantages of multiple restriction enzymes for cloning

[velvetvelour]

why would utilizing duo restriction enzymes for cloning be better then just one?
( e.coli to pBluescript II )

 

[Monique]

That way the vector can't self-ligate.

 

[velvetvelour]

thanks a lot Monique

 

[Ygggdrasil]

You can prevent self-ligation fairly effectively by pretreating the cut vector with a phosphatase. One major advantage of using two restriction enzymes for cloning is that you can control the orientation that your DNA fragment is inserted into the vector (especially useful if you're making a construct to express protein).