- T4 DNA ligase.
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- Description
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- 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 ãnicksÒ in DNA duplexes, RNA or DNA/RNA hybrids,
and the interconversion of ATP and AMP by the exhange of pyrophosphate
groups.
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- Unit definition
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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.
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- Reaction buffers
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- B14 (x10)
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- Reaction conditions
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- 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.
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- Storage
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- Store at -20°C.
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- Applications
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- construction of recombinant DNA molecules; detection of nicked DNA,
joining RNA single strands via bridging oligonucleotide adapters.
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Quality control
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activity, SDS-PAGE/purity, absence of RNase and DNase activities, mock
cloning assay.
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References
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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.
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- Catalog #
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- M6.4
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M6.5
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Pack size
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1.000 U
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2.500 U
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