| Product |
T4
DNA Ligase
- MBI |
| Description |
Purified from an E.coli starin carrying
a plasmid with the cloned gene encoding this enzyme.
T4 DNA Ligase catalyzes the formation of a phosphodiester
bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini
in duplex DNA or RNA. |
| Storage |
-20°C |
| Applications |
Joining double-stranded
DNA with cohesive or blunt termini.
Joining of oligonucleotide linkers or adaptors to blunt-ended
DNA.
Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids. |
| Note |
· One Weiss unit
is equivalent to approximately 200 cohesive-end ligation
units. One cohesive-end ligation unit is defined as the
amount of enzyme required to give 50% ligation of HindIII
fragments of Lambda DNA in 30min at 16°C in 20µl
of the assay mixture (50mM Tris-HCl, pH 7.5), 10mM MgCl2,
10mM DTT, 1mM ATP, 25µg/ml BSA and a 5'-DNA termini
concentration of 0.12µM (300µg/ml). The ratio
of Weiss unit to cohesive-end ligation unit is determined
by conversion of [5'-33P]-labeled termini of HindIII fragments
of Lambda DNA to a phosphates-resistant form.
·
T4DNA ligase is strongly inhibited by NaCl or KCl if the
concentration exceeds 200mM.
· The volume of
the ligation reaction mixture, the concentration of the
DNA, the amount of enzyme, the temperature and incubation
time depends on the application.
· Polyethylene
glycol (PEG) greatly increases the rate of ligation of
blunt-ended DNA. The final recommended concentration of
PEG 4000 in the reaction mixture is 5%(W/V).
·
The inactivation of the T4 DNA Ligase by heating at 65°C
for 10 minutes is recommended as a standard procedure
prior to transformation of cells with DNA. In some cases
this simple step can increase the number of transformants
by two orders of magnitude. |
|
