Bio-Rad Ligation and Transformation Module - Manual (Page 23)

Products of Ligation
Ligation is a very inefficient process; from millions of vectors and inserts,
only 1–100 are expected to ligate together as desired and lead to growth
of colonies from the desired transformants. There are several possible
products from a ligation with a vector such as the pJet1.2 blunted vector:
• Self-ligation of the vector — A self-ligated vector, without any DNA
inserted in the MCS, should have an intact lethal gene. The product of
the lethal gene should kill any bacteria transformed by these vector-only
constructs. (Note: The eco47IR gene is not 100% lethal, and so some
bacteria may grow, resulting in tiny satellite colonies, which may be
observed in no-insert ligation controls)
• Ligation of a vector with primer-dimers or other short DNA fragments
from the PCR reaction — Even if these fragments are a small proportion
of the total fragments available for ligation, small fragments ligate more
easily than larger fragments
• Ligation of a vector with multiple inserts — Since the inserts all have
the same blunt ends, they can ligate to each other and also ligate to
the vector, giving a product with multiple inserts in a row. The number
of these products can be reduced by controlling the molar ratio of insert
to vector. If the ratio is high (that is, if there are many more insert
molecules than vector), then it is more probable that inserts will ligate
to each other rather than ligate to the vector. So the molar ratio can be
an important factor in setting up ligation reactions
• Self-ligation of insert — It is possible for the insert molecules to
self-ligate, forming closed circles. Since these molecules do not have
any of the vector genes, they will not be able to replicate, so they are
not of consequence
• Ligation of one insert into a vector — This is the desired result, and in
ligation to a vector such as pJet1.2 blunted vector, there is no
directionality in the cloning. In other words, the insert can ligate into the
vector in either direction, because all of the ends are identical. So the
products should be a 50:50 mix, with half of the inserts in one
orientation and the other half in the reverse orientation
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