222 Maundershigher concentrations favor intermolecular reaction (oligomerization
and formation of recombinant molecules). Condensing agents exert a
similar effect. For example, 10% polyethylene glycol enhances inter-
molecular ligation (54,55). Longer DNA molecules are more prone to
recircularization than shorter ones.
Optimal ratios usually have to be determined empirically, though
they are fairly broad, and a 30% variation in starting concentrations
makes little difference. Some theoretical guidelines have been sug-
gested by Revie et al. (56) for vectors of 2.5-7.5 kb and inserts of 0.2-
10 kb, which can be briefly summarized as follows;
- Always use forced directional (asymmetric) cloning and phosphatased
vectors where possible. - Keep the vector DNA concentration below 1 ~g/mL, unless "scaveng-
ing" for minute quantities of insert DNA. - Under normal conditions, use 3:1 molar ratio of insert:vector, but not
more than 5 lag/mL insert DNA. - Increase the insert concentration as the vector size decreases.
In practice, a suitable preliminary experiment would use a concen-
tration of 20-60 lag/mL vector (2-3 kb in size) with an equimolar or
slightly greater concentration of insert DNA (47).
The above rules apply only to plasmid ligations. For the formation
of~, or cosmid concatamers, insert and vector should be ligated at a 1:1
molar ratio in as high a concentration as possible.
In all cases, the ability to select for the recombinant DNA molecule
of choice by, for example, transformation and expression of an antibi-
otic resistance or by enzymic formation of a colored product from a
chromogenic substrate would offer much more flexibility in the choice
of DNA concentrations and reaction conditions of the ligation.
3.6. Reaction Protocol
A specific example of a ligation reaction protocol for DNA frag-
ments with cohesive termini is as follows:
3.6.1. Materials Required- l0 mM Tris-HC1 / 1 mM EDTA, pH 7.5.
- Phenol.
- Chloroform.
- Ethanol.