Question about Restriction enzyme - (Apr/17/2007 )
I am engineerng a enzyme site inside my gene and i selected some but every one has its different capacity to cut and ligate.
I want to know about enzyme comments after 10- fold overdigestion with some enzyme > 95 percent can be ligate and recut.
what this mean after 10- fold overdigestion. some enzyme are after 200 fold overdigestion. whats this concept of fold overdigestion.
This has to do with how many units and hours of digestion are performed, and the amount of DNA. 1 unit nominally cuts 1 ug in 1 hour, so a 10 fold overdigestion would use 10 units cutting 1 ug for 1 hour. I never pay much attention to this, and use 20-100x overdigestion routinely. The 95% comment is not quite right. What it says is slightly but importantly different -- that after digestion and religation, 95% of the ligated DNA can be recut. This does not tell you an important number -- the fraction of DNA which is successfully cut. This is, in my experience, closer to 80%, and the remainder is quite resistant to cutting. But it sounds as if you are trying to choose enzymes. Since there is a fair amount of (knowledge, lore, rumor, prejudice) about enzymes, you might ask around about other people's experiences (like here, for example) about the enzymes you plan on using.
Thanks for very good and important information
Supposing you have 2 enzymes, A is 10 fold over digestion and B 200 fold overdigestion.
Now if you digest a sample separately with A and B using same number of units and everything.
the sample with enzyme B can be left for a 20 fold longer digestion time compared to enzyme A which after some time will start to damage the digested ends.
Enzyme with lower fold digestions can easily damage the sticky ends and cause problems with ligations. So if you were to digest with such enzymes, take care how long you incubate them.