Quick Ligase

Index Size
EN12-050 50 reaction
EN12-150 150 reaction

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Product Features

Quick Ligase is an ATP-dependent recombinant enzyme, isolated from Escherichia coli, used for DNA cloning in just 5 to 15 minutes. Quick Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. It will join both blunt-ended and cohesive-ended restriction fragments of DNA as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

 

Applications

- Ligation in just 5-15 minutes

- Cloning of PCR products

- Cloning of restriction fragments

- Joining of double-stranded oligonucleotide linkers or adaptors to DNA

- Site-directed mutagenesis

- Nick repair in duplex DNA, RNA or DNA/RNA hybrids

- Self-circularization of linear DNA

- LM PCR methods (Ligation Mediated PCR), eg. Amplified fragment length polymorphism (AFLP)

 

Enzyme Concentration

5 U/µl

 

Storage Buffer

10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM KCl, 50% (v/v) glycerol

 

2x Quick Ligation Buffer

132 mM Tris-HCl (pH 7.6), 20 mM DTT, 20 mM MgCl2,15% PEG 6000

The 2x Quick Ligation Buffer does not contain ATP, which must be added separately. For most applications, ATP should be added to the reaction to a final concentration of 0.5-1.0 mM. The 25 mM ATP Solution is included.

 

Inhibition and Inactivation

heat inactivated at 65°C for 10 minutes or 70°C for 5 minutes

 

Unit Definition

One (Weiss) unit of Quick Ligase catalyzes the conversion of 1 nmol of 32P from pyrophosphate into Norit-adsorbable material in 20 minutes at 37°C.

 

Quality Control

Quick Ligase is functionally tested in cloning assays and is free of detectable contaminating nuclease activities. The following QC assays are performed on each new lot:

 

- Endonuclease Activity (Nicking) assays

1 µg of supercoiled pUC19 plasmid DNA is incubated with 50 U of Quick Ligase in 1× Quick Ligation Buffer for4 hours at 37°C. Following incubation, the DNA is visualized on an agarose gel. There must be no visible nicking or cutting of the DNA.

 

- Non-Specific DNase Activity assay

The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

 

- Functional assay

Linearized pUC19 plasmid (leaving either blunt-end or cohesive ends) is re-ligated with 5 U of Quick Ligase. The DNA is then transformed into E. coli competent cells that are plated on ampicillin plates. The re-ligation efficiency is determined by counting transformed bacterial colonies.

 

Shipping Conditions

Quick Ligase is shipped on dry or blue ice.

 

Storage and Stability

All components should be stored at -20°C. When stored under optimum conditions, the reagents are stable for a minimum of 12 months from date of purchase.

 

References

1. Weiss, B. et al., Enzymatic breakage and joining of deoxyribonucleic acid. J. Biol. Chem. 243, 4543-4555 (1968).

2. Pheiffer, B.H. et al., 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).

3. 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).

4. Nilsson, M. et al., RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 29, 578-581 (2001).

5. 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).

 


Manufacturer: BLIRT S.A.