Dephosphorylation: Incomplete Dephosphorylation or Incomplete Digestion?
Dephosphorylation of vector ends facilitates ligation with inserts decreasing self-ligation. When a large number of blue colonies appear on a plate, one might think the dephosphorylation would be incomplete. In my experience however, this is not due to incomplete dephosphorylation but incomplete restriction digestion which is almost unavoidable.
Commercially available competent E. coli shows transformation efficiency of about 108 cfu/ug pUC19. When 10pg of plasmid is transformed to 100uL of such cell solution, plating of 100uL of 10 times dilution of over-grown cells in SOC yields about 100 transformants. In case of 99.9% digestion of 100ng vector, uncut plasmid amounts to 100pg! In this case up to 1000 blue colonies would appear. Such small amount of uncut plasmid DNA is of course undetectable on a gel stained with EtBr, so we cannot see incomplete digestion. Very weak signal is however, detectable at the position expected for cc form when Southern blotted.
The second round of digestion is employed to achieve complete digestion. Routinely mini-prepped plasmid is however, difficult to be cut completely even if recut. Some inhibitory effects are suspected such as partial denaturation to single strand or existence of inhibitors against restriction enzymes. In addition from my experience, termini of enzyme-cut vector become difficult to ligate upon repeated enzyme treatments or freeze-thaw cycles possibly because of degradation. It is therefore better to reduce number of steps from restriction to ligation. High transformation efficiencies of plasmid containing inserts were achieved when these procedures were done within a day.
We should then eliminate uncut vector in another way. Cutting out from an agarose gel is the most effective and easiest. Because preparation of insert DNAs is generally cutting out from an agarose gel, loading the vector on the same gel is rather convenient. Comparing fluorescent intensity of bands, we can adjust the best vector-insert ratio, mix two gel slices in one tube, and extract DNAs using a commercial kit (GeneClean; BIO101, USA).
Reaction condition of dephosphorylation is not critical. For 5'-protrusion or blunt ends, add 1uL (c.a.20U) of CIP to the restriction digestion mixture. Digestion and dephosphorylation are accomplished at the same time. For 3'-protrusion ends, add additional 1uL of CIP after the above reaction and incubate 30 min more at 50oC. These procedures were successful for routinely used buffers for restriction enzymes (M, H, K, and T buffers). I have not tried for L buffer.
Efficiency is different among enzymes from different suppliers. CIP from Toyobo was not effective when stored for 3 years. The storage solution of the enzyme does not contain Zn. CIP from Takara with the storage solution containing Zn is effective when stored for more than 3 years. Zinc as a co-factor of CIP may be related to this difference.
