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Agarase

#EO0461	100 u (0.5 u/µl)
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EO0461 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Gentle recovery of intact DNA (see Fig.1) and RNA.
• High yield recovery of DNA compared to other extraction methods (see Fig.2).
• Efficient recovery of long DNA fragments (>30 kb).
• Active in various electrophoresis buffers.
• The recovered nucleic acids may be directly used for sequencing, amplification, etc.

Description
The Agarase specifically digests the agarose polysaccharide core made up of repeating 1,3-linked beta-D-galactopyranose and 1,4-linked 3,6-anhydro-alfa-L-galactopyranose into neoagaro-oligosaccharides (1).

Source
E.coli cells with a plasmid containing the gene encoding beta-agarase from Pseudomonas atlantica.

Molecular Weight
32.7 kDa monomer.

Application
Quantitative recovery of DNA or RNA from low melting point (LM) agarose gels, see Protocol for the Recovery of DNA from Low Melting Point Agarose Gels.

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases, ribonucleases and phosphatases confirmed by appropriate quality tests. Functionally tested for recovery of DNA.

Concentration
0.5 u/µl

Definition of Activity Unit
One unit of the enzyme completely degrades 100 µl (approx. 100 mg) of molten 1% low melting point agarose in 30 min at 42°C.
Enzyme activity is assayed in the 1X TBE buffer: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA.

Storage Buffer
The enzyme is supplied in: 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

Inhibition and Inactivation

• Inhibitors: ethidium bromide at concentrations higher than 5 µg/ml, transition metal ions.
• Inactivated by heating at 70°C for 10 min.

Note

• Incubate at 42°C.
• Activity of Agarase in different buffers (in comparison to activity in assay buffer (1X TBE)):
  • 1X TBE (90 mM Tris-borate, 2 mM EDTA, pH 8.3) 100%
  • 1X TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.5) 120%
  • 1X TPE (90 mM Tris-phosphate, 2 mM EDTA, pH 7.7) 120%
  • 1X Bis-Tris (50 mM Bis-Tris-HCl, 1 mM EDTA, pH 6.5) 150%
• Conventional agarose is not suitable for digestion by Agarase.

Figure 1. Recovery of DNA fragments from low melting point agarose gel using Agarase.
The GeneRuler™ 1 kb DNA Ladder  fragments (lane M) were extracted from 1% TopVision™ LM GQ Agarose gel.
Sizes of DNA fragments (lanes 1-14): 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 750, 500, 250 bp.

Figure 2. Superior DNA recovery with Agarase.
Different extraction methods were used to extract DNA (0.5-10 kb) from TopVision™ LM GQ Agarose.

Related Products

TopVision™ LM GQ Agarose InsTAclone™ PCR Cloning Kit CloneJET™ PCR Cloning Kit T4 DNA Ligase T4 RNA Ligase Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit Glycogen Water, nuclease-free

References

1. Yaphe, W., The use of agarase from Pseudomonas atlantica in the identification of agar in marine algae (Rhodophyceae), Can. J. Microbiol., 3, 987-993, 1957.
2. Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips Online, T40022, 1996.
3. Adkins, S., Burmeister,M., Visualization of DNA in agarose gels and educational demonstrations, Anal. Biochem., 240 (1), 17-23, 1996.

Updated vasario 05, 2008 15:26