General Notes
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. T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes. 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 -20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used. Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1 µg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/µg) for 4 hours at 37°C released <0.3% of the total radioactivity. Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1 µg of X174 RF I DNA for 4 hours at 37°C resulted in <5% conversion to RFII as determined by agarose gel electrophoresis. 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.
