220 MaundersAfter reaction, the enzyme can be inactivated at 65°C for 10 min or
by addition of dimethyl dicarbonate to 0.1% and heating to 37°C for
10 min. The DNA can then be concentrated by ethanol precipitation,
which also removes magnesium ions that may reduce the efficiency of
a subsequent transformation step. If ethanol precipitation is not per-
formed, the magnesium concentration should be reduced by fivefold
(or greater) dilution of the ligation mixture.
Ligation can be achieved directly on DNA in low melting agarose
excised from a gel, though more enzyme is required and the efficiency
is 10-fold lower. The gel should be run in TAE buffer (40 mMTris-acetate,
1 mM EDTA, pH 7.5-8.0) rather than the more usual Tris-borate-
EDTA (TBE). The presence of ethidium bromide in the gel at 0.5 lag/mL
causes no ill effects (47).
E. coli DNA ligase will function in a similar buffer to T4 DNA ligase,
although the Tris-HC1 concentration is usually reduced (20-30 mM)
and ATP replaced by 25-50 ~4 NAD. DTT (20 mM) is often included
in the buffer, although this may be superfluous (37). In some cases, 10 mM
ammonium sulfate and 10 mM KC1 are also added.
3.4. Reaction Conditions--Blunt Termini
The ligation of blunt- or flush-ended DNAs is only possible using
T4 DNA ligase and not E. coli DNA ligase (19,49). The reaction is
slower than the ligation of cohesive ends, but is extremely useful in
that it can be used to join virtually any two pieces of DNA, be they
naturally occurring or synthetic.
In practice, this ability can be made universal by the infilling or "pol-
ishing" of cohesive or incompatible termini. This technique can be
adapted to infill partially "ragged" termini leaving short cohesive termini
that are not self-ligatable, but that will accept inserted DNA (50,51).
Blunt-end ligation can be performed in the standard buffer with the
ATP concentration adjusted to 0.5 mM (52). More ligase should be
used, of the order of 50 U/mL and 1-2 U/~xg DNA. Optional additions
for blunt-end ligation include 100 I.tg/mL BSA, 1 mMhexamine cobalt
chloride, and 0.1-1.0 mM spermidine. The latter two reagents can
increase the efficiency of linker ligation by fivefold.
The DNA concentration should also be raised. In the case of small
oligomers, such as linkers, it is easy to achieve a high concentration
of DNA termini of the order of 4-20 laM or up to 100-fold in excess