Certificate of Analysis and Data Sheet

Source:
E. Coli. lambda lysogen NM 989

Catalog No.
ENZ-286

Description :

Recombinant T4 DNA Ligase 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.

Physical Appearance:

Sterile filtered liquid formulation having a concentration of 10,000 U/ml.

Formulation:

T4 DNA Ligase is supplied in 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50% glycerol.

Reaction Conditions:

50 mM Tris-HCl (pH 7.8 @ 25⁰C), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA and DNA (0.1 to 1 μM in 5?termini). Οptimal ligation occurs at 16⁰C.

Stability:

Two years when stored at ?0蚓, 2 weeks at 4蚓.

Unit Definition:

1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of λ DNA (5?DNA termini concentration of 0.12 然, 300- ug/ml) in a total reaction volume of 20 μl in 30 minutes at 16蚓 in 1X T4 DNA Ligase Reaction Buffer.
2. One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of 32P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37蚓.

Activity:

One Weiss unit is equivalent to circa 67 cohesive-end ligation units.
?T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration is > 200mM.
?Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt-end ligation may be enhanced by addition of PEG 4000 (10% w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50 μM.
?To dilute T4 DNA Ligase that will subsequently be stored at ?0⁰C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.

Heat Inactivation:

T4 DNA Ligase can be inactivated by incubation at 65⁰C for 10 minutes.

Quality Control:

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.

Exonuclease Activity:

Incubation of a 50μl reaction containing 13,000 units of T4 DNA Ligase with 1μg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/μg) for 4 hours at 37慢 released < 0.3% of the total radioactivity.

Endonuclease Activity:

Incubation of a 50μl reaction containing 13,000 units of T4 DNA Ligase with 1μg of ΦX174 RF I DNA for 4 hours at 37慢 resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis.

Nuclease Activity:

Incubation of 13,000 units for 18 hours in assay buffer (without ATP) with Hind III fragments of λ DNA yielded a clear and sharp banding pattern on agarose gels.

Applications:

?Cloning of restriction fragments.
?Joining linkers and adapters to blunt-ended DNA

Usage:

This material is offered by ProSpec-TechnoGene for research, laboratory or further evaluation purposes.

Latest Publications:

1. Template-independent ligation of single-stranded DNA by T4 DNA ligase.
FEBS J 2005 Dec;272(23):5991-6000
2. Addendum: Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase.
Biotechniques 2005 Sep;39(3):328
3. Activity-based in vitro selection of T4 DNA ligase.
Biochem Biophys Res Commun 2005 Oct 28;336(3):987-93
4. Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase.
Biotechniques 2005 Jun;38(6):864, 866, 868
5. Improved full-length cDNA production based on RNA tagging by T4 DNA ligase.
Nucleic Acids Res 2004 Jan 2;32(1):e6
6. Ligase-mediated construction of branched DNA strands: a novel DNA joining activity catalyzed by T4 DNA ligase.
Nucleic Acids Res 2004 Jan 2;32(1):e2