protein

Recombinant T4 DNA Ligase

MBS142427

20000 IU

160 USD

Recombinant Protein

Recombinant T4 DNA Ligase

T4 DNA Ligase

T4 DNA; T4 DNA Ligase Recombinant; DNA ligase 4; EC 6.5.1.1; DNA ligase IV; Polydeoxyribonucleotide synthase [ATP] 4

T4 DNA

Escherichia Colilambda lysogen NM 989

50mM Tris-HCl (pH 7.8 at 25 degree 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 16C. Sterile filtered liquid formulation having a concentration of 167,000 U/ml.

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

Exonuclease Activity: Incubation of a 50 ul reaction containing 13,000 units of T4 DNA Ligase with 1 ug of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/ug) for 4 hours at 37 degree C released < 0.3% of the total radioactivity. Endonuclease Activity: Incubation of a 50 ul reaction containing 13,000 units of T4 DNA Ligase with 1 ug of X174 RF I DNA for 4 hours at 37 degree C resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis. Heat Inactivation: T4 DNA Ligase can be inactivated by incubation at 65C for 10 minutes. 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. Unit Defenition: 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- ug/ml) in a total reaction volume of 20 ul in 30 minutes at 16 degree 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 degree C. Storage Buffer: 50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 ug/ml BSA and 50% glycerol. Store at -20C.

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

ENZYMES; Enzymes; Ligase

Description: 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.

20000 IU

160 USD

100000 IU

345

500000 IU

1165