What techniques can be used to clone an insert into a lentiviral vector containing only one restriction site?
If a lentiviral vector contains only one restriction site, one can use standard cloning techniques to ligate the insert into this site. If it is not immediately feasible to digest and clone the insert from a parent vector, some possible approaches to using this site include subcloning or appending compatible restriction sites onto the insert of interest using PCR. The process of subcloning consists of digesting the insert of interest from its parent vector into a second vector in such as way that the insert may later be digested from this new vector and cloned into the lentiviral vector (this is basically shuffling restriction sites between vectors until the gene of interest is flanked by sites compatible with those in the vector into which one ultimately want to ligate the insert). Often times it is less time consuming and easier to simply add restriction sites onto the insert of interest using PCR. This is accomplished by PCR amplifying the insert sequence using primers that contain
