I have a plasmid with RE sites that produce blunt and sticky ends. I need to do cloning on this. Is it possible to ligate both ends simultaneously together in one reaction?
yes, you will have no problem getting a clone. Not only that but you will know its orientation. Cloning with blunt ends only can be tricky, in this case its straight forward.
Good luck...
Thanks Gary for quick reply. So does that means I can ligate my gene of interest easily if the gene has one blunt and other sticky end ? Any special protocol ?
No special protocol, the vector is cut with the sticky and blunt cutting enzymes, followed by dephosphorylation and your favourite clean up (Phenol extraction + precipitation, or column purification with a kit). The fragment (carrying your gene of interest) is cut with a compatible sticky cutting enzyme, and any blunt cutting enzyme, and it is gel-purified. Then you do 3 ligations:
1) vector alone without ligase, (test for uncut plasmid)
2) vector alone with ligase, (test for self-ligation)
3) vector + fragment with ligase (this is where tyou expect most colonies after transformation).
Self-ligation happens when the dephosphorylation is not 100% complete. The clean up does not completely remove the smkall polylinker fragment you have removed (and that is invisible). It also happens that a very small (invisible) portion of the plasmid vector has only been cut by one of the two enzymes and because dephosphorylation is never 100% complete you can then get a lot of self-ligation. Intramolecular ligations are much mor efficient than intermolecular ligations (two DNA fragments finding each other and ligating occasionally).
Check the compatibility of the RE enzyme buffers to determine if a double digest is possible first. If they are not then I would digest with the weaker enzyme first, purify, then use the other RE. It is not necessary to dephosphorylate the plasmid as you have non compatible sites (blunt and 5') and so long as the RE enzymes are working well the background Intramolecular recombination will be very low. I allays purify using column kits as this avoids the issue of phenol or ethanol carryover and loss during precipitation (also its much faster). Finally I ligate overnight at 4oC.
Hope that helps
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