I am trying to clone a 700 bp fragment to a 7 kb vector. My insert has ClaI restriction site in both ends and my vector has 1 ClaI accordingly. I have been told to use CIP in the linearized vector to remove the phosphates in order to avoid vector self-ligation when I try for the ligation, yet I wonder how can a ligation of insert and vector occur if I remove the phosphates from the vector, why the ligation of insert is not sensitive to the vector not having a phosphate if the T4 enzyme is phosphate group dependent? how is exactly the dependence of the T4 ligase working?
Inigo, majority of RE live 5' P and no P on 3' of the produced ends. So if you remove P from you vector than it will be ligated at reduced ( not zero) rates. Your fragment still caries 5' P and thus will ligate with P-less 3' ends of your vector, leaving fragment-3' strand, vector 5' strand non ligated, This ss-break will be repaired by bacteria through plasmid amplification ( bacterial grows). Non-ligated linear vectors will be lost. Hope it make sense.
Yes, thanks! additionally, is there something I can make during the ligation possibly to reduce the possibility of having two inserts (or more) in the vector?
Iñigo, I would advise you to use SAP (shrimp alkaline phospatase) instead of CIP. This last is quite difficult to eliminate from the reaction, and a carry-over of residual CIP with the vector will contaminate the ligation reaction, dephosphorylating the insert and result in a low yield of recombinant clones. SAP is killed just by heating....
If you don't want to clone multiple copies of insert just adjust the vector:insert ratio to 1:1 (molar ratio). Allowing an extra time to the ligation reaction (e.g. overnight) should compensate the reduction in insert.
Good luck and let us know whether you suceed or have aditional problems.
alright, thanks a lot, I checked and we indeed have a rSAP in the lab so ill use it. I think I will try the 1:1 ratio as you suggest, and if I have enough material left also the 1:3 which has always worked fine for me, might be interesting to have the control. Lets see what happens!
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