Applications
- Ligation of blunt or cohesive-ended DNA fragments.
- Repair of nicks in double-stranded nucleic acids.1
T4 DNA Ligase is an ATP-dependent ligase used for DNA cloning. It covalently
joins dsDNA molecules having 5´-phosphorylated and 3´-hydroxylated
blunt or compatible cohesive ends produced by restriction enzyme digestion
or other enzymatic processes.2,3 T4 DNA Ligase has no activity
on single-stranded nucleic acids. Following a ligation reaction, T4 DNA Ligase
may be inactivated by incubation at 65°C for 10 minutes.
For most cloning applications, we recommend the Fast-Link™ DNA Ligation
Kits because they are optimized for rapid ligations with extremely high ligation
efficiencies.
Unit Definition: One Weiss unit of T4 DNA Ligase converts 1 nmol
of 32P from pyrophosphate into Norit-adsorbable material in 20
minutes under standard assay conditions. One Weiss unit equals approximately
67 Cohesive-End Units.
Note: T4 DNA Ligase is available at concentrations of 2 Weiss
Units/µl (134 Cohesive End Units/µl), and at a higher 10 Weiss
Units/µl (670 Cohesive End Units/µl) for blunt-end ligation.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5),
0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.
T4 DNA Ligase 10X Reaction Buffer: 330 mM Tris-acetate (pH 7.8),
660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT. The Reaction
Buffer does not contain ATP, which must be added to the reaction to a final
concentration of 0.5-1.0 mM. A 25-mM solution of ATP is included.
Quality Control: T4 DNA Ligase is function-tested in cloning assays
and is free of detectable contaminating DNA exo- and endonuclease and RNase
activities. |
Figure 1. Ligation activity of EPICENTRE's T4 DNA Ligase. T4 DNA
Ligase at 10 U/µl was diluted sequentially in 10-fold increments. One
microliter of each dilution of T4 DNA Ligase was incubated with Hind
III-cut lambda DNA at 16°C in 1X Reaction Buffer containing ATP. Lane
1, kb ladder; Lane 2, no enzyme; Lane 3, 1 U/µl; Lane 4, 0.1 U/µl;
Lane 5, 0.01 U/µl; Lane 6, 0.001 U/µl; Lane 7, 0.0001 U/µl;
Lane 8, 0.00001 U/µl. Note that full enzyme activity is seen down to
one one-thousandth unit; enzyme activity is still apparent even at one ten-thousandth
unit. |
