I am looking to amplify a gene from a separate plasmid and insert into a plasmid. the plasmid i will cut with a restriction enzyme that gives sticky ends! Do I need to use T4 polymerase to blunt these ends before the gibson assembly reaction?
No, it does not need to be blunt. But you do need to understand what's left of your sticky ends after T5 nuclease is done with it. Draw the sticky ends in double-stranded notation, and then visualize how T5 nuclease is going to chew one strand back, but not the other.
Then, you can use NEBuilder to check on your understanding. It does it automatically.
The sticky ends in double-stranded notation, and then visualize how T5 nuclease is going to chew one strand back, but not the other.
It is not mandatory to have vector with blunt ends. I am going to perform Gibson Assembly and before that I have simulate it on Snapgene software with sticky end vector, and the software gave me a green signal.
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