Description
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal 5ʹ phosphate and a 3' hydroxyl group of duplex DNA or RNA. The enzyme efficiently joins blunt and cohesive ends and repairs single-stranded nicks in duplex DNA, RNA or DNA:RNA hybrids (1). This enzyme is supplied in 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol: pH 7.5 at 25°C. The 10x T4 DNA Ligase Buffer contains 500 mM Tris-HCI, 100 mM MgCl 2, 50 mM DTT and 10 mM ATP; pH 7.6 at 25°C. The 2x Rapid Ligation Buffer contains 132 mM Tris-HCl, 20 mM MgCl 2, 2 mM DTT, 2 mM ATP and 15% PEG 6000; pH 7.6 at 25⁰C.
Storage temperature: –25°C to –15°C Molecular weight: 55,292 Daltons Test Units tested Specification Purity n/a >99% Specific activity n/a 300,000 U/mg Single-stranded exonuclease 6000 U <1% released Double-stranded exonuclease 6000 U <1% released Double-stranded endonuclease 6000 U No conversion E. coli DNA contamination 6000 U <10 copies
The recombinant enzyme protein is produced by a recombinant E. coli strain carrying the cloned T4 DNA Ligase gene. One unit is defined as the amount of T4 DNA Ligase required to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µL 1x T4 DNA Ligase Buffer following a 30 minute incubation at 23°C.
Usage Instructions For reaction setup for T4 DNA Ligase (150,000 U) and T4 DNA Ligase (240,000 U). Reagent Amount Final Concentration 10x T4 DNA Ligation Buffer 2 µL Variable Vector Variable 1–10 ng/µL Insert Variable 1–10 ng/µL T4 DNA Ligase (120 U/ µL) 1 µL 6 U/µL Type I Water Variable n/a Total volume 20 µaL n/a 1. Transfer all components listed in the table above to a clean reaction vessel, and mix well by pipetting. 2. Incubate at 25°C for 30 minutes. 3. Immediately purify DNA using PCR clean-up columns and elute in approximately 50 µL. 4. Alternatively, dilute (at least 1:10, but ensure that 0.1–10 ng ligation product is available for transformation) immediately in TE or water. 5. Transform 0.1–10 ng ligation product into a chemically or electrocompetent cell line compatible with the vector. Notes One T4 DNA Ligase cohesive end unit is equivalent to approximately 3 cohesive end units as measured with a Lambda-Hind III DNA fragment substrate in 1x T4 DNA Ligase reaction buffer. One Weiss Unit is approximately equivalent to 22 T4 DNA Ligase cohesive end units. T4 DNA Ligase is ATP dependent. We recommended that the reaction buffer be discarded after one year of storage at –20°C and replaced with fresh buffer to ensure maximum performance. Single-insert ligations are optimal when targeting an insert: vector ratio between 2 and 6. A ratio above 6:1 will promote the insertion of multiple fragments, while a ratio below 2:1 will reduce ligation efficiency. For problematic ligations or if the DNA concentration is unknown, it may be necessary to vary ratios and run multiple ligations. The 10x T4 DNA Ligase Buffer does not contain PEG and is compatible with standard ligation protocols that do not specify the use of a rapid, fast or quick format buffer. Best: Purify the product using a DNA purification spin column and elute in 50 µL of TE following ligation. The DNA is now ready for a transformation. The final amount of DNA to be transformed should be in the range of 0.1–10 ng. Better: Dilute ligation product in ddH20 or TE to reduce the PEG concentration. The final amount of DNA to be transformed should be in the range of 0.1–10 ng. High-concentration T4 DNA Ligase, in combination with the 2x Rapid Ligation buffer, greatly stimulates the rate and efficiency of blunt-end ligation; therefore, long incubations (>10 minutes) are NOT recommended and can significantly reduce the transformation efficiency of ligation products. The following protocol is recommended to maximize the transformation efficiency of the correct insert/vector combination. The 10x T4 DNA Ligase Buffer does not contain PEG and is compatible with standard ligation protocols, which do not specify the use of a rapid format buffer. Reference 1. Engler, M.J., and Richardson, C.C. (1982) P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press. Quality Control Unit activity was measured using a two-fold serial dilution method. The enzyme was diluted in 1x T4 DNA Ligase Reaction Buffer and added to 20 µL reactions containing double-DNA fragments and 1x T4 DNA Ligase Reaction Buffer. Reactions were incubated for 30 minutes at 23°C, stopped, and analyzed on a 1% agarose gel stained with ethidium bromide. Protein concentration is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
