T4 DNA ligase.
Description
T4 DNA Ligase (EC 6.5.1.1) catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA. The enzyme will not join ss nucleic acids. Detected activities include the joining of hydrogen-bonded cohesive DNA termini (with either 5´ or 3´ projecting single-stranded regions), the joining of blunt-ended double-stranded DNA molecules, the resealing of single-stranded „nicks“ in DNA duplexes, RNA or DNA/RNA hybrids, and the interconversion of ATP and AMP by the exhange of pyrophosphate groups.
Unit definition

1 Weiss unit catalyzes the exhange of 1 nmol of 32P from inorganic pyrophosphate to ATP in 20 min at 37°C. 0,01 Weiss units ligate >95% of the Hind III fragments from 1 µg lambda DNA in 30 min at 16°C.

Reaction buffers
B14 (x10)
Reaction conditions
50 mM TrisHCl 7.6, 10 mM MgCl2, 10 mM DTT, 0,5-1 mM ATP, 50 µg/ml BSA (optional),0,1 Weiss U/µg DNA. The enzyme has an optimum temperature for ligating cohesive ends of 4°C, for sealing nicks of 37°C and for blunt-end ligation of 25°C (if DNA fragments >16 bp long; smaller molecules requiring lower temperatures). Similar results for ligation of cohesive ends might be achieved by incubation at 25°C for 1 h, at 15°C for 4-6 h, and at 4°C for 16 h. The maximum effect for blunt-end ligation is achieved by the addition of PEG8000 to 15%, MgCl2 to 5 mM and ATP to 0,5 mM. Similar effect is observed by addition of BSA to 100 µg/ml, hexammine cobalt chloride to 1.0-1.5 mM and spermidine to 0,1-1 mM. Blunt-end ligations require 10 to 100 times more enzyme than cohesive-end ligations to achieve an equal efficiency.
Storage
Store at -20°C.
Applications
construction of recombinant DNA molecules; detection of nicked DNA, joining RNA single strands via bridging oligonucleotide adapters.

Quality control

activity, SDS-PAGE/purity, absence of RNase and DNase activities, mock cloning assay.

References

1. Weiss, B. et al. (1968) J. Biol. Chem. 243, 4543. 2. Modrich, P. et al. (1972) J. Biol. Chem. 247, 6370-6372. 3. Murray, N.E. et al. (1979) J. Mol. Biol. 132, 493-505.

Catalog #
M6.4

M6.5

 

 

 

Pack size

1.000 U

2.500 U

 

 

 

 

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