|Products||beta-Agarase from USB Corp.|
|Description|| Source: Pseudomonas atlantica|
Beta-Agarase is useful in recovering DNA and RNA fragments from low-melting-temperature agarose. Beta-Agarase digests the agarose into oligo- and disaccharides.
Tested User Friendly
Heat Inactivation: 65C, 15 min
Molecular Weight: 30,000 Da
Optimum pH: 6.5
Tested for contaminating endonucleases, exonucleases and ribonucleases.
50mM Bis-Tris-HCl (pH 6.5), 1mM EDTA, 50% glycerol.
Shipping and Storage:
Shipped on dry ice. Store at -20C.
Reaction mixture contains 10mM Bis-Tris-HCI (pH 6.5) and 1mM EDTA.
One unit is the amount of enzyme required to digest 200 l of melted low-melting-point agarose to oligo- and disaccharides in 1 hr at 40C.
Tested User FriendlyTM Functional Test:
Isolation of linearized plasmid from low-melting agarose, > 50% recovery.
Quantitative recovery of DNA from low-melting point agarose gels.
PROTOCOL FOR RECOVERING FRAGMENTS FROM LOW-MELT AGAROSE
*Equilibrate DNA/agarose in at least 2 volumes of 1X Beta-Agarase Reaction Buffer for approximately 20 min.
*Spin briefly and remove the supernatant.
*Incubate DNA/agarose at 70C for 10 min to melt the agarose.
*Cool to 40C and maintain the temperature at 37C.
*Add an equal volume of 1X Beta-Agarase Reaction Buffer.
*Add one unit of Beta-Agarase for every 50 l of agarose gel. Do not include the volume of the buffer for this determination.
*Incubate the enzyme/DNA/agarose for 1-18 hours at 37C. The treated DNA may be used directly for ligation, restriction digests, etc. or further purified as in steps 8-12.
*Extract once with buffer equilibrated phenol.
*Extract once with buffer equilibrated phenol:chloroform (1:1).
*Extract twice with chloroform:isoamyl alcohol (24:1).
*Add 2 volumes of TE (pH 7.6) to prevent precipitation of the oligosaccharides. Precipitate with 0.05 volumes of NaCl and 2 volumes of 95% ethanol. Incubate at -20C for 30 min, then spin in a microcentrifuge. Wash the pellet twice with 70% ethanol.
*The DNA solution may be used directly for ligation, restriction digests, transformation and sequencing or the purified DNA may be concentrated.
*YAPHE, W. (1957) Can. J. Microbiol. 3, 987-993.
*BURMEISTER, M. AND LEHRACH, H. (1986) Nature 324, 582-585.
Functionally Tested 10X Beta-Agarase Reaction Buffer (1 ml included, PN 73432):
100mM Bis-Tris-HCl (pH 6.5), 10mM EDTA
|Info|| USB Corp.|
26111 Miles Road
Cleveland Ohio 44128
Fax Number: 216.464.5075
Web Site: http://www.usbweb.com