Ligases 219
- 1-5 mM DTT
- 2OO lag/mL BSA
- 50% Glycerol
High concentrations of enzyme are very stable at -20°C. If diluted
to 500 U/mL, the enzyme is 90% stable at this temperature for 6 mo.
Lower concentrations are increasingly less stable. For example, a 10
U/mL dilution will lose 40% of its activity in 3 mo. Storage at tempera-
tures lower than -20°C may also be detrimental.
3.3. Reaction Conditions--Cohesive Termini
Reaction conditions can vary with the purity of each batch of enzyme,
the purity of the DNA substrate, and the presence of buffer compo-
nents from any previous or subsequent in vitro reaction step. DNA
ligase acts in virtually all restriction enzyme buffers and also in the
same buffer used to kinase linkers prior to their ligation (47).
A commonly used buffer for ligation reactions with T4 DNA ligase
is given below with a range of acceptable parameters:
20 mM Tris-HC1 pH 7.6
10 mM MgC12
10 mMDTT
1 mM ATP
0.1 Weiss units enzyme(20-66 mM, pH 7.5-7.8)
(5-50 mM)
(5-20 mM)
(66 !.O4- 1 mM)
(0.01-1.0 U/lag DNA)The inclusion of BSA (Fraction V) at 50 lag/mL is optional.
DNA concentration (see Section 3.5.):
Reaction vol 20 ~tL (10-100 lxL)
Temperature 16°C (4-25°C)
Time 4 h (20 min-24 h)
If DNA substrates with cohesive ends are being ligated, the DNA
should be heated to 70°C for 10 min to melt any hydrogen bonding
between the termini of similar molecules before the various DNA
substrates are mixed. After heating, the mixture should be allowed to
cool slowly to allow intermolecular reannealing, prior to the addition
of the reaction buffer and finally the DNA ligase.
Reaction times and temperatures have an inverse relationship. For
example, similar results might be achieved by incubation at 25°C for
1 h, at 15°C for 4-6 h, and at 4°C for 16 h (48).