T4 DNA Ligase (T4 DNA) Active Protein

Escherichia Colilambda lysogen NM 989

(167,000 U/ml) 50mM Tris-HCl (pH 7.8 at 25°C), 10mM MgCl₂, 10mM DTT, 1mM ATP, 25 ug/ml BSA and DNA (0.1 to 1 um in 5´ termini).

Optimal ligation occurs at 16°C.

Two years when stored at -20°C, 2 weeks at 4°C.

Protein activity can be an ambiguous term. Please refer to the data sheet or inquire with Technical Support to determine the definition of activity for this product.

Sterile filtered liquid formulation

50mM KCI, 10mM Tris-HCI (pH 7.4),0.1 mM EDTA, 1mM DTT, 200 ug/ml BSA and 50% glycerol. Store at -20°C.

1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of DNA (5´ DNA termini concentration of 0.12 uM, 300- µg/ml) in a total reaction volume of 20 ul in 30 minutes at 16°C 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°C.

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 50uM.
• To dilute T4 DNA Ligase that will subsequently be stored at –20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.

T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes.

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/ug) for 4 hours at 37°C 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 X174 RF I DNA for 4 hours at 37°C 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 gamma DNA yielded a clear and sharp banding pattern on agarose gels.

Enzymes; Ligase