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DNA Modifying Enzymes and Cloning >
Ligases >
T4 DNA Ligase |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).
Source:
Purified from E. coli C600 pcl857 pPLc28 lig8 (2).
Applications:- Cloning of restriction fragments
- Joining linkers and adapters to blunt-ended DNA
Reagents Supplied:
T4 DNA Ligase Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
65°C for 10 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X T4 DNA Ligase Reaction Buffer
Incubate at
16°C.
1X T4 DNA Ligase Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM Dithiothreitol
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.
Concentration:
400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
- To dilute T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A, NEB #B8001S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
- Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP.
Application notes:- Room Temperature Ligation:
For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (NEB #M2200S, [30 reactions] or NEB #M2200L, [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.
FAQs
- What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
- What are some other problems that should be considered when trouble shooting a transformation problem?
- What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail?
- What controls should be run to test the cells and DNA when using T4 DNA Ligase?
- When should T4 DNA Ligase be the enzyme of choice?
- Can the T4 DNA Ligase be used with the Quick Ligase buffer?
- How much DNA should be used in a ligation using T4 DNA Ligase?
- Can T4 DNA Ligase be used in other NEBuffers?
- Can T4 DNA Ligase be heat inactivated?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases. Each lot of T4 DNA ligase is also tested in a mock cloning assay which reveals any damage to the ligated DNA termini. Greater than 99.9% of the termini remain undamaged in this assay.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 3,200 units of T4 DNA Ligase with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Nuclease Activity:
Incubation of 3,200 units for 18 hours in assay buffer with HindIII fragments of λ DNA yielded a clear and sharp banding pattern on agarose gels.
Exonuclease Activity:
Incubation of 3,200 units of enzyme with 1 μg sonicated 3H DNA (105 cpm/μg) for 4 hours at 37ºC in 50 μl Exonuclease III Reaction Buffer released < 0.1% radioactivity.
Figure 1: Ligation of HindIII fragments (4-base overhang) of lambda DNA using 1 unit (1 µl of a 1:400 dilution) of T4 DNA ligase. Incubation at 25°C.
Figure 2: Ligation of blunt-ended HaeIII fragments of Lambda DNA using various amounts of T4 DNA ligase in a 20 µl reaction volume. Incubated for 30 minutes at 16°C.
References
- Engler, M.J. and Richardson, C.C. (1982) P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press.
- Remaut, E., Tsao, H. and Fiers, W. (1983) Gene, 22, 103-113.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd Ed.), 1.53-1.73.
Reagents Sold Separately
T4 DNA Ligase Reaction Buffer
Companion Products
Quick Blunting™ Kit
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