Dear All,
I want to linearize a plasmid by SacII, which have one restriction site for SacII. Should I keep it for 3-4hr at 37°C for getting at least 90% efficiency? Suggest me any better idea.
Thank You.
As you know SacII requires 2 sites in order to get efficient cleavage. It is my understanding that plasmids with a single site are cleaved only very very inefficiently, so I'm not sure how well you will get the plasmid cleaved under those conditions.
You could consider adding a second SacII site nearby through site directed mutagenesis or by ligating an oligo with a SacII site into an adjacent restriction site.
You can also activate the SacII by using a double stranded oligo with a SacII site in trans, but you have to get the ratios very accurate in order for this work.
see this:
https://www.neb.com/en/tools-and-resources/usage-guidelines/restriction-enzymes-requiring-multi-sites-for-efficient-cleavage