Bio-Rad Ligation and Transformation Module User Manual
Page 20
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One advantage to sticky-end ligation is that it makes directional
cloning possible. If it is desirable to have the insert in one orientation
only (for instance, in the A
Æ B direction in the vector, but not in the
B
Æ A direction), then the insert and vector can both be digested with two
different restriction enzymes so that their ends are asymmetric. This is important
if the DNA insert is a cDNA encoding a protein to be expressed in the
transformed cell. When this is done, only the complementary ends will ligate
and the insert will have a single orientation in the ligation products.
Blunt end digestion.
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