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T4 DNA Ligases

T4 DNA Ligase
#EL0015 #EL0014 Both supplied with: 10X Ligation Buffer	200 u (1 u/µl) 200 u (5 u/µl) 0.5 ml
#EL0016 #EL0011 #EL0017 All supplied with: 10X Ligation Buffer	5x200 u (1 u/µl) 1000 u (5 u/µl) HC, 1000 u (30 u/µl) 1.5 ml
#EL0012 #EL0013 Both supplied with: 10X Ligation Buffer	5x1000 u (5 u/µl) HC, 5000 u (30 u/µl) 5x1.5 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EL0011, #EL0012,  #EL0013, #EL0014, #EL0015, #EL0016, #EL0017 MSDS (English) MSDS (English-USA) MSDS (German)
T4 DNA Ligase (with PEG)
#EL0335 #EL0334 Both supplied with: 10X Ligation Buffer 50% PEG Solution	200 u (1 u/µl) 200 u (5 u/µl) 0.5 ml 0.5 ml
#EL0336 #EL0331 #EL0337 All supplied with: 10X Ligation Buffer 50% PEG Solution	5x200 u (1 u/µl) 1000 u (5 u/µl) HC, 1000 u (30 u/µl) 1.5 ml 1.5 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EL0331, #EL0334,  #EL0335, #EL0336, #EL0337 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Active in Fermentas buffers for restriction enzymes, PCR and RT, supplemented with ATP.

Description
The T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA with blunt or cohesive-end termini. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids but has no activity on single-stranded nucleic acids (1, 2).
The T4 DNA Ligase requires ATP as cofactor.

Source
E.coli cells with a cloned gene 30 of bacteriophage T4.

Molecular Weight
55.3 kDa monomer.

Applications

• Joining double-stranded DNA with cohesive or blunt termini (3, 4), see protocols for DNA Insert Ligation into Vector DNA, for Self-circularization of Linear DNA.
• Joining of oligonucleotide linkers or adaptors to blunt-ended DNA (3, 4), see protocol  for Linker Ligation.
• Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids (5).
• Ligase-mediated RNA detection (6).
• Site-directed mutagenesis (7).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests. Functionally tested for the capacity to join cohesive- and blunt-ended DNA fragments.

Concentration
1 u/µl, 
5 u/µl,
30 u/µl, HC

Definition of Activity Unit
One unit of the enzyme catalyzes the conversion of 1 nanomole of [³²PP_i] into Norit-adsorbable form in 20 min at 37°C (Weiss unit) (8).
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 30 min at 16°C in 20 µl of the assay mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 µg/ml BSA and a 5'-DNA termini concentration of 0.12 µM (300 µg/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 phosphatase-resistant form.
Enzyme activity is assayed in the following mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 0.066 mM ATP, 10 mM DTT, 3.3 µM [³²PP_i].

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 50 mM KCl, 0.1 mM EDTA and 50% (v/v) glycerol.

10X Ligation Buffer
400 mM Tris-HCl, 100 mM MgCl₂, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C).

Inhibition and Inactivation

• Inhibitors: T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM.
• Inactivated by heating at 65°C for 10 min.

Note

• Polyethylene glycol (PEG) greatly increases the rate of ligation of blunt-ended DNA (9). 5% (w/v) is the suggested concentration of PEG 4000 in the reaction mixture.   
• The T4 DNA Ligase binding to DNA may result in a band shift in agarose gels. To avoid this, prior to electrophoresis we recommend to incubate the samples with Fermentas 6X Loading Dye & SDS Solution (#R1151) at 65°C for 10 min and chill on ice.
• It is necessary to remove the enzyme from the ligation mixture by chloroform extraction prior to electro-transformation of bacterial cells with DNA. 

Related Products

Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit CloneJET™ PCR Cloning Kit InsTAclone™ PCR Cloning Kit TransformAid™ Bacterial Transformation Kit Nb.Bpu10I T7 DNA Polymerase T4 DNA Polymerase Hot Start Taq DNA Polymerase TrueStart™ Taq DNA Polymerase Pfu DNA Polymerase Taq DNA Polymerase (native) Taq DNA Polymerase (recombinant) M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase First Strand cDNA Synthesis Kit RevertAid™ First Strand cDNA Synthesis Kit RevertAid™ H Minus First Strand cDNA Synthesis Kit Calf Intestine Alkaline Phosphatase (CIAP) Shrimp Alkaline Phosphatase (SAP) 6X Loading Dye & SDS Solution ATP Water, nuclease-free

References

1. Rossi, R., et al., Functional characterization of the T4 DNA Ligase: a new insight into the mechanism of action, Nucleic Acids Res., 25, 2106-2113, 1997.
2. Cherepanov, A.V., et al., Binding of nucleotides by T4 DNA Ligase and T4 RNA Ligase: optical absorbance and fluorescence studies, Biophys. J., 81, 3545-3559, 2001.
3. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
4. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.
5. Engler, M.J., Richardson, C.C., DNA ligases, The Enzymes (Boyer, P.D., ed.), Academic Press Inc., San Diego, vol.15B, 3-30, 1982.
6. Nilsson, M., et al., RNA-templated DNA ligation for transcript analysis, Nucleic Acids Res., 29, 578-581, 2001.
7. Cherepanov, A., et al., Joining of short DNA oligonucleotides with base pair mismatches by T4 DNA Ligase, J. Biochem., 129, 61-68, 2001.
8. Weiss, B., et al., Enzymatic breakage and joining of deoxyribonucleic acid, J. Biol. Chem., 243, 4543-4555, 1968.
9. Pheiffer, B.H., Zimmerman, S.B., Polymer-stimulated ligation: enchanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA ligase in polymer solutions, Nucleic Acids Res., 11, 7853-7871, 1983.

Updated kovo 18, 2008 10:04