Definition of Unit (for specific activity): 1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of DNA (5Â´ DNA termini concentration of 0.12 µM, 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.
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
Additional Information: 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.
Bioactivity Data: 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 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 50µM. To dilute T4 DNA Ligase that will subsequently be stored at Â20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.
Reconstitution Instructions: |||
Buffer: 50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 µg/ml BSA and 50% glycerol. Store at -20C.
Physical Appearance: Sterile filtered liquid formulation having a concentration of 167,000 U/ml.
Limitations and Performance Guarantee
enQuire Bio's products are guaranteed and approved for RESEARCH USE ONLY or manufacturing purposes. This product may not be used as a pharmaceutical agent, or other product intended for human consumption without meeting additional regulatory requirements. This product is guaranteed to work for a period of two years when stored at -70C or colder, and one year when aliquoted and stored at -20C.