Question: Ligating and Cutting at the same time - (Jan/21/2006 )

I now have read a lot about performing a ligation and a restriction at the same time to prevent self-ligations. And since I am a curious person I want to try that method now. But first I have some questions:

1.I want to ligate a blunt end fragment (EcoRV) into a pBluescript-Vector. If I add EcoRV wouldn't that not only cut the self-ligated vectors but also the wanted ligations?

2.I am using a 20 μl sample. The T4-DNA Ligase is from Roche and the ligation is performed o/n at 4°C. How much restriction enzyme should I use. And is it necessary to add a restriction-buffer? I got controversal answers when I tried to google it up.


Thanks for the answers!

On your first question: you will cut your wanted ligations if your PCR product (or whatever it is you want to ligate) also has been cut with EcoRV. (or if is is blunt end PCR product in whish the primers have part of the EcoRV sequence).

for the buffers: you better first check their composition or you can cut a vector with EcoRV in ligation buffer.

I have successfully used one-phor-all buffer (Amersham) in doing restriction digests and ligations in the same tube at the same time. Depending on which restriction enzyme you are using, you use either 1X or 2X final buffer concentration. The fact sheet you get with the buffer has this information.