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Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation

l for the Rapid DNA Ligation Kit that was still suboptimal with respect to DNA concentration and recommended dephosphorylation of the vector DNA, the yield of transformants was 5-fold higher than with the optimized conventional ligation procedure. This remarkably high efficiency of the kit is most likely due to an optimized composition of the ligation buffer.

Although, in theory, 100% correct clones can be obtained by a strategy involving fragments that are designed to join in the desired order and orientation, a notable number of false-positive clones arose in all our ligation experiments. Most of them were due to contamination resulting from incompletely cleaved and self-ligated phage DNA backbone. A relatively small proportion of deletion mutants and clones containing a doubled phage backbone was also found.

The excellent performance of the Rapid DNA Ligation Kit was reproducible with different insert pairs. Thus, the kit provides a reliable and remarkably efficient way for the three-fragment assembly DNA ligation to generate circular DNA under minimal time requirements.


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