Cloning vector

[roniadam]

Hi everyone:

how many enzymes are required to open a cloning vector for insertion of the cloned gene? I am using PFastBac vector and I cut it by one enzyme only. Is this will allow the interested fragment to insert into the vector or I have to use two enzymes???

[canalon]

1 enzyme is enough but the risk is religation of the vector without integration of the fragment. And of course you wil not be able to select the orientation of your insert, which can be a problem if you plan to express a protein.