|Products||Ligase, T4 DNA from USB Corp.|
|Item||Ligase, T4 DNA|
|Description|| Source: E. coli strain containing an overproducing clone of T4 DNA Ligase.|
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed
5' phosphoryl and 3'-hydroxyl termini in duplex DNA or RNA. It repairs single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids and will join both blunt-end and cohesive-end fragments of duplex DNA or RNA(1).
Tested User Friendly
Molecular Weight: 55,000 Da
Optimum pH: 7.5 - 7.8
Optimum Temperature: 16C for exchange reaction
Requirements for Divalent Cation: Mg2+, Mn2+
Optimum Mg2+ Concentration: 10mM
Sulfhydryl Requirement: 2-mercaptoethanol, dithiothreitol
Stimulators: Spermine (0.5 to 1mM), Spermidine (0.5 to 1mM)
Inhibitors: Na (>0.2M), K (>0.2M)
Inactivation: Heat at 65C for 10 minutes
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, exonucleases and ribonucleases.
25mM Tris-HCl (pH 7.6), 100mM NaCl, 1mM DTT, 0.5mM EDTA, 50% glycerol.
Shipping and Storage:
Shipped on dry ice. Store at -20C.
T4 DNA Ligase is assayed according to the pyrophosphate exchange method of Weiss
et al. The reaction mixture (300 l) contains 67mM Tris-HCl, (pH 7.8), 6.7mM MgCl2, 10mM DTT, 66M ATP and 3.3M radiolabeled pyrophosphate. Incubation is at 37C for
Note: ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA Ligase which requires NAD.
One Weiss unit is the amount of enzyme required to convert 1 nmol of radiolabeled phosphate from pyrophosphate into absorbable material in 20 min at 37C under standard assay conditions. One Weiss unit equals about 67 cohesive end ligation units.
PN 70005: 1 unit/l; PN 70042: greater than or equal to 5 units/l
Tested User FriendlyTM Functional Test:
Re-ligation of linearized plasmid DNA >75% of control, as determined by counting transformed bacterial colonies.
*Ligation of cohesive-end or blunt-end termini.
*Ligation of synthetic linkers or adaptors.
PROTOCOL FOR DNA LIGATION
For each 10 l of ligation reaction, a combination of 2040 ng of vector DNA and a 1:3-10 fold molar excess of foreign DNA will produce an adequate yield of recombinant genomes for
most cloning purposes.
1. Combine the following:
Vector DNA (2040 ng) __ l
Insert DNA (310-fold molar excess) __ l
Ligation Buffer, 10X 5 l
Water __ l
T4 DNA Ligase __ l (1 unit)
Total 50 l
2. Mix and incubate at 16C for 16 hrs.
Note: Km for the activity of T4 DNA Ligase on blunt-ended DNA is nearly 100 times higher than that on DNA with cohesive ends. Therefore, ligation of blunt-ended DNA should be carried out at a higher concentration of both vector and insert DNA.
To stimulate the blunt-end ligation reaction, 150200mM NaCl and 5% PEG can be added.
*ROSSI, R., MONTECUCCO, A., CIARROCHI, G. AND BIAMONTI, G. (1997) Nucl. Acids Res. 25 (11), 2106-2113.
*WEISS, B., JACQUEMIN-SABLON, A., LIVE, T. R., FAREED, G. C. AND RICHARDSON, C. C. (1968) J. Biol. Chem. 243, 4543-4555.
Functionally Tested 10X T4 DNA Ligase Reaction Buffer (1 ml included, PN 70087): 660mM Tris-HCl (pH 7.6), 66mM MgCl2, 100mM DTT, 660M ATP
T4 DNA Ligase Dilution Buffer (1 ml included, PN 70108): 20mM Tris-HCl (pH 7.6), 60mM KCl, 5mM DTT, 1mM EDTA, 50% glycerol
|Info|| USB Corp.|
26111 Miles Road
Cleveland Ohio 44128
Fax Number: 216.464.5075
Web Site: http://www.usbweb.com