DESCRIPTION
T4 DNA ligase is isolated from a recombinant E. coli strain and is suitable for ligation of sticky- and blunt ended DNA fragments. The enzyme repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids, joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.
The T4 DNA Ligase requires ATP as cofactor.

APLICATIONS
Joining double-stranded DNA with cohesive or blunt termini
Joining of double-stranded oligonucleotide linkers or adaptors to double-stranded DNA
Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids
Ligase-mediated RNA detection
Site-directed mutagenesis
Amplified fragment length polymorphism

CONCENTRATION
10 Weiss units/µl

UNIT DEFINITION
One unit (Weiss unit) is the amount of enzyme that catalyses the conversation of one nanomol 32P-ATP in 20 minutes at 37°C. 1 ligation unit = 0,015 Weiss unit.

STORAGE BUFFER
20 mM Tris-HCI buffer pH 7.5; 100 mM NaCI; 0.1 mM EDTA; 2 mM DTT; 0.1 mg/ml BSA; 50% glycerol (v/v)

STORAGE TEMPERATURE
Store T4 DNA ligase below 0ºC, preferably at -20ºC, in a constant temperature freezer.

10X REACTION BUFER
660 mM Tris-HCI pH 7.5; 50 mM MgCI₂; 10 mM DTE; 10 mM ATP
+ extra 1 ml 50% PEG4000 solution on request

REACTION CONDITION
optimal ligation occurs at 15ºC




NOTES
PEG (Polyehtylenglycol) greatly increases the rate of ligation of blunt-ended DNA (1). 5% (w/v) is the suggested concentration of PEG4000 in the reaction mixture.
Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 min or 65°C for 10 min and chill on ice prior to electrophoresis.
The volume of the ligation reaction mixture should not exceed 10% of the competent cell volume in the transformation process.
Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture by spin column or chloroform extraction. The extracted DNA can be further precipitated with ethanol.

REFERENCES
1. Pheiffer, B.H., Zimmermann, S.B., Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA Ligase in polymer solutions, Nucleic Acids Res., 11, 7853-7871, 1983