Hi, I have a vector that has AvrII (at 5' end) and XhoI(at 3' end) RE sites at the cloning site for my insert. I wish to know if these two enzymes give rise to overhangs that are complementary or would self-ligate instead of ligating to the insert, thereby producing vector self-ligated colonies.
AvrII: 5'-CCTAGG-3'
XhoI: 5'-CTCGAG-3'
Hello, no you do not have a risk of self-ligation, the 2 enzymes do not generate complementary ends, they are not compatible enzymes.
AgeI would give Cohesive end and SmaI blunt end. Can a double digest my vector and insert using AgeI and SmaI and ligate the respective end?
yes, you have the risk of self-ligating your vector when digested with AgeI and XmaI, because these 2 enzymes generate complementary ends. You don't have this risk when digesting with AgeI and SmaI since they don't generate complementary ends.
However, be careful if one of the enzymes does not work you will have the risk of opening the vector with a single enzyme and there you will have the risk of self-ligation.
I'm opting for AgeI+ SmaI double digestion.. but one generates cohesive end another blunt
Will that ligate in a same reaction with respective ends on my insert?
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