DNA ligase from Rhodothermus marinus is a thermostable NAD+ dependent double-stranded DNA ligase with broad temperatures range between 5 and 65°C. The enzyme catalyzes ligation of DNA with high fidelity and is suitable for various applications including gene synthesis.

Product Dlig120 Rma DNA ligase is available as 500 units and 2000 units together with 10x reaction buffers.

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Product description

Introduction:

Rma DNA ligase catalyzes the NAD-dependent ligation of adjacent 3´-hydroxyl and 5´-phosphate termini in duplex DNA structures. In contrast to T4 DNA ligase, Rma DNA ligase has no detectable activity on blunt end DNA fragments. Unlike T4 DNA ligase, Rma DNA ligase shows only minimal ligation activity under optimal temperature conditions for 4 bp as well as 2 bp of co-hesive ends. Rma DNA ligase has no activity on RNA targets. Rma DNA ligase is isolated and purified from an E.coli strain carrying a plasmid with the cloned DNA ligase gene from the thermophilic bacteria Rhodother-mus marinus isolated in Iceland (1, 2). The half-life of Rma ligase is 7 min at 91°C (2). The enzyme has a broad range of reaction temperatures with the optimal activity about 55°C. Under optimal conditions the rate and extent of oligonucleotide ligation is higher for Rma DNA ligase compared to Tth and Taq DNA ligases (3,4).

Applications:

Tsc DNA ligase is an ideal enzyme for applications requiring high temperature, high-stringency ligations of double-stranded DNA.Tsc DNA ligase may be applied to:

Gene Synthesis (12) from overlapping oligonucleo-tides.
 

Storage:
Storage and dilution buffer: 20 mM Tris-HCl, 50 mM KCl, 0,1 mM EDTA, 0,1% Triton X-100 (v/v), 1 mM dithiothreitol (DTT), 50% glycerol (v/v), pH 7,6 (25°C). Rma DNA ligase is stable when stored at –15°C to -25°C.

Reaction conditions:
1 x reaction buffer (10 x supplied) 20 mM Tris-HCl, 20 mM KCl, 10 mM MgCl2, 0,1% Nonidet P40 (v/v), 0,5 mM NAD, 1 mM DTT, pH 7,5 (25°C).

Concentration and unit definition
Concentration 10 U/μl. One unit of Rma DNA ligase catalyzes the ligation of 50% of the cos sites of 1μg BstEII digested λDNA in 1 min at 45°C.

Application Protocol:

Activity assay:
The enzyme assay for unit definition was ligation of cos sites of λDNA digested with BstII.

Component Volume Final conc.
Reaction buffer (10x) 2,0 μl 1 x
λDNA (BstEII digested) X μl 1 μg
Tsc DNA Ligase Dilution serie 
Add sterile H2O Up to 20,0 μl 
TOTAL 20,0 μl 
Incubate at 45°C for 1-15 min.Stop reaction in dry ice/ethanol bath. Incubate for 10 min at 65°C before analysis on agarose gel (melting of not ligated cos sites)

Results are assayed by agarose gel electrophore-sis and ethidium bromide staining.

Quality Control:

Quality Control:
Each lot of Tsc DNA Ligase is assayed for activity and for contaminating activities as stated below.

Absence of DNA endonuclease:

0,25 μg supercoiled pBR322 DNA is incubated with increasing amounts of Tsc DNA ligase in 25 μl reactions at 37°C for 16 h. >100 U of Tsc DNA ligase show no relaxation of the supercoiled structure of pBR322 DNA.
0,25 μg of λ-DNA Eco RI/HindIII fragments is incu-bated with Tsc DNA ligase in 25 μl reactions at 37°C and 64°C for 16 h. 100 U of Tsc DNA ligase show no alteration of the banding pattern.
Absence of exonuclease:
Increasing amounts of Tsc DNA ligase are incubated in 50 μl test buffer containing [3H]-labelled DNA at 37°C and 64°C for 4 h. The amount of enzyme, which shows no exonuclease activity is >100 U.

Absence of Rnases:
RNaseAlertTM Lab Test Kit (cat no. 1964) from Ambion was used to detect RNase activity according to the manufacturer protocol. No RNase activity was detected after incubating X U of Tsc DNA ligase at 37°C after 1 hours.