Hi, i am an undergraduate student and i'm trying to clone a gene fragment in a plasmid vector for the fist time for me. I have some questions, one of them is that i was reading that in some protocols gives the advice that if you want to insert a PCR product it most have be phosforilated before the ligation step, so you have to use a kinase to achieve that, but i also understand that restriction enzymes (at least some of them) leave a 5P end. So when i digest my PCR product it will be enough to leave 5P ends? Thanks in advance for your answers.
I concur with XI Jiang; in addition even if you were trying to clone a PCR fragment by blunt ligation ( no restriction cutting of the fragment), the 5’ phosphate provided by restriction cutting of the vector would be sufficient: the ligated product would carry a single-strand nick, but the latter would be fixed in vivo after transformation
- Badges
- Science method