T4 DNA Protein

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 50μM.• 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.

Applications: Cloning of restriction fragments.Joining linkers and adapters to blunt-ended DNA. Unit Definition: 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 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. Note: 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. 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. Storage Buffer: 50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 μg/ml BSA and 50% glycerol. Store at -20C. Inactivation: T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes. 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. 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

50mM Tris-HCl (pH 7.8 at 25°C), 10mM MgCl2, 10mM DTT, 1mM ATP, 25 μg/ml rAlbumin and DNA (0.1 to 1 μm in 5 ́ termini) . Optimal ligation occurs at 16C.