E. coli DNA Ligase

Description

E. coli DNA Ligase catalyzes the formation of a phosphodiester bond between the 5´-phosphate and the 3´-hydroxyl of two adjacent DNA strands in duplex DNA with cohesive ends. It is not appreciably active on blunt-ended substrates. E. coli DNA Ligase uses NAD as a cofactor and can be heat-inactivated. E. coli DNA Ligase is active at a range of temperatures (4° C – 37° C). 

Highlights

  • Specific Activity: 6,000 units/mg
  • Supplied with 10X Reaction Buffer

Product Source

Purified from E. coli strain containing a cloned E. coli DNA Ligase gene.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
E. coli DNA Ligase Reaction Buffer-2010X

Advantages and Features

Applications

  • Okayama and Berg cDNA cloning (3)

Properties and Usage

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 E. coli DNA Ligase Reaction Buffer.

Reaction Conditions

1X E. coli DNA Ligase Reaction Buffer
Incubate at 16°C

1X E. coli DNA Ligase Reaction Buffer:
30 mM Tris-HCl
4 mM MgCl2
26 μM NAD
1 mM DTT
50 μg/ml BSA
pH 8 @ 25°C

Usage Concentration

10,000 units/ml

Storage Temperature

-20°C

Storage Conditions

300 mM NaCl
10 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
500 μg/ml BSA
50% Glycerol
200 μg/ml BSA
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Notes

  1. Requires NAD+ (nicotinamide adenine dinucleotide) as a cofactor, in contrast to other ligases which use rATP.
  2. Ligation of blunt-ended fragments is extremely inefficient. For ligation of blunt-ended fragments use T4 DNA Ligase.
  3. Does not ligate RNA to DNA (4).
  4. This enzyme ligates only DNA fragments with cohesive termini.

References

  1. Panasenko, S.M. et al. (1977). Science. 196, 188-189.
  2. Panasenko, S.M. et al. (1978). J. Biol. Chem.. 253, 4590-4592.
  3. Okayama, H. and Berg, P. (1982). Mol. Cell. Biol.. 2, 161-170.
  4. Higgins, N.P. and Cozzarelli, N.R. (1979). R. Wu(Ed.), Methods Enzymol.. 68, 50-71. New York: Academic Press.

FAQs

  1. How does E. coli DNA Ligase (NEB# M0205) differ from T4 DNA Ligase (NEB# M0202)?
  2. Which ligase should I use?
  3. Will E. coli DNA Ligase work in CutSmart Buffer?

Protocols

  1. E. coli DNA Ligase Protocol (M0205)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour, DNA):
    The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.