T4 DNA Ligase

• Efficiently joins blunt and cohesive ends
• Repairs single-stranded nicks in duplex DNA, RNA and DNA:RNA hybrids
• Choice ligase for sequencing workflows

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal 5ʹ phosphate and a 3' hydroxyl groups 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.

10X T4 DNA Ligase Buffer (cat. no. B6030) contains the following: 500 mM Tris-HCI, 100 mM MgCl₂, 50 mM DTT and 10 mM ATP; pH 7.6 at 25°C.

2X Rapid Ligation Buffer (cat. no. B1010) contains the following: 132 mM Tris-HCl, 20 mM MgCl₂, 2 mM DTT, 2 mM ATP and 15% PEG 6000; pH 7.6 @ 25⁰C.

5X Rapid Ligation Buffer (cat. no. B9020) contains the following: 330 mM Tris-HCl, 50 mM MgCl₂, 5 mM DTT, 5 mM ATP and 30% PEG 6000; pH 7.6 @ 25⁰C.

SDS available on request.

Enzyme properties

• Storage temperature: –25°C to –15°C

OEM by QIAGEN offers bulk manufacturing of T4 DNA Ligase in custom formulations.

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.

Unit definition: 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.

Molecular weight: 55,292 Daltons

Instructions for reaction setup for T4 DNA Ligase (150,000 U), cat. no. L6030-LC-L, and T4 DNA Ligase (240,000 U), cat. no. L6030-HC-L.

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 ensuring 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 that is 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. Enzymatics 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 or fast or quick format buffer.

Best: Following ligation, purify the product using a DNA purification spin column and elute in 50 µL of TE. The DNA is now ready for 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.

Enzymatics’ high-concentration T4 DNA Ligase in combination with the 2X Rapid Ligation buffer greatly stimulates the rate and efficiency blunt-end ligation, therefore long incubations (>10 minutes) are NOT recommended and can greatly reduce the transformation efficiency of ligation products. To maximize transformation efficiency of the correct insert/vector combination, the following protocol is recommended.

Enzymatics 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/fast/quick 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.

Instructions for T4 DNA Ligase (144,000 U), cat. no. L6030-W-L

1. Transfer Y µl of DNA adapter (see note below) into the PCR tube with 50 µl of A-tailed DNA following 5X WGS fragmentation (cat. no. Y9410L) or 5X ER/A-tailing (cat. no. Y9420L). Mix gently by pipetting and then place on ice.
2. Prepare the following ligation reaction master mix (for each DNA sample) in a separate tube on ice and mix well by pipetting. The master mix can be scaled as needed for the desired number of samples (10% extra volume added to compensate for the pipetting loss when preparing the master mix for multiple samples).

   1 reaction (µl)
   5X Ligation Buffer	20
   DNA ligase	10
   Nuclease-free H2O	(20 - Y)
   Total	(50 – Y)
3. Add (50 – Y) µl of the ligation master mix to the sample from step 1 and mix well by pipetting. Incubate the ligation reaction at 20°C for 15 minutes.
   IMPORTANT: Do not use a thermocycler with a heated lid.
4. Proceed immediately to adapter ligation cleanup using 0.8X (80 µl) AMPure XP beads.

a) Equilibrate AMPure XP beads to room temperature for 20 minutes.
b) Add 80 µl of thoroughly vortexed AMPure XP beads slurry to the ligation sample from step 3 and mix well by pipetting.
c) Incubate the mixture for 5 minutes at room temperature. Pellet the beads on a magnetic stand (e.g., DynaMag) and carefully discard the supernatant.
d) Wash the beads with 200 µl 80% ethanol. Pellet the beads on the magnetic stand and discard the supernatant. Repeat the wash once.
e) Air-dry the beads on the magnetic stand for 10 minutes or until the beads are dry. Over-drying of beads may result in lower DNA recovery.
f) Resuspend the dried beads in 52.5 µl 10 mM Tris-HCl; pH 8.0. Pellet the beads on the magnetic stand. Carefully transfer 50 µl supernatant into a new tube.

5. If no size selection is required, perform a second purification using 1X (50 µl) AMPure XP beads. Elute DNA in 28 µl 10 mM Tris-HCl; pH 8.0. Pellet beads and carefully collect 25.5 µl purified DNA sample for library amplification. If size selection is required, please use your choice of method and follow the corresponding protocols. Alternatively, if library amplification is not intended, elute DNA in 12.5 µl 10 mM Tris-HCl, pH 8.0, after the second 1X AMPure XP beads purification. Pellet beads and carefully collect 10 µl of purified DNA sample. If not proceeding immediately, the sample can be stored at –20°C.

* Note: DNA adapters are not included. Follow supplier’s recommendation for adapter concentration and usage condition. Generally, we recommend an adapter to insert molar ratio from 25:1 to 200:1 based on the amount of input DNA (1µg – 1ng) and size of the targeted DNA fragment to achieve optimal ligation efficiency.

• Quality control analysis

Unit activity was measured using a-twofold serial dilution method. Dilutions of enzyme were made 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 OD₂₈₀ 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 mass of the band 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.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Restriction cloning
• TA cloning
• Adapter ligation
• NGS library construction and cloning