Hello
I am new to cloning and repating an experiment.
I read on promega that i need to dephosphorylate vectors when i am subcloning. Should i take the gene away from 1st vector and put the insert into the second vector first and then do the dephosporilation and run on gel?
or should i insert the gene into the second vector and run on gel, then choose the band of interest, gel purify and then dephosphorylate?
How can i be sure the dephosporilation was succesfull?
Thank you!
Dephosphorylation of the vector is done if you want to insert a fragment into a vector and need to prevent re-ligation of the vector without any insert. So you dephosphorylate after you have cut the vector, then purify the cut vector before adding the insert.
If your dephosphorylation did not work, you might get a lot of religated vectors without any insert, resulting after transfection in clones that contain the vector, but not the gene of interest.
Both answers are correct. I would only add one extra thing. You should only dephosphorylate your linear vector if you digested it with ONE restriction enzyme. If you used TWO enzymes that don't have compatible ends you don't need to dephosphorylate.
Thanks a lot for the valuable answers!
Dear Andre I will be using 2 RE but they create 5nt sticky ends. The first nt doesnt match but the other 4nts can anneal with each other. Despite the 1nt being incompatible, I assume they will be able to ligate with the other 4nts?
I would still need dephospoylation right?
Can you say the name of the enzymes so I can take a look at them?
Sener don't you worry! BamHI and HindIII can't self ligate. I've done it lots of time. The extremities are not compatible. As long as both enzymes work properly you don't need to dephosphorylate your vector.
Elena, are you referring to needing to dephosphorylate the inserts? If so, then no.
Dephosphorylation is only necessary for the vector backbone. You are simply trying to prevent the backbone closing on itself and giving you colonies that can propagate this empty vector (absent of your gene of interest).
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