Storage Condition

−20℃

Components

Note: 1 U = 1 Weiss unit

Description

T4 DNA Ligase (Fast) is expressed in E. coli carrying the T4 bacteriophage gene 30. It catalyzes phosphodiester bond formation between 5′-phosphate and 3′-hydroxyl groups of dsDNA or RNA. It repairs nicks in dsDNA, RNA, or DNA/RNA hybrids and ligates sticky- or blunt-end fragments. It requires ATP and completes sticky-end ligations at room temperature in just 10 minutes.

Applications include cloning of digested fragments, site-directed mutagenesis, PCR product cloning, linear DNA circularization, and dsDNA nick repair.

Definition of Activity Unit

1 Weiss unit converts 1 nmol of [³²P]PPi into Norit-adsorbable form in 20 min at 37℃. One Weiss unit ≈ 200 cohesive end ligation units (CEU), corresponding to ligation of λDNA HindIII fragments in 30 min at 16℃.

Quality Control Assays

• Endonuclease Activity: <10% nicked/linearized after 4 h with 200 ng plasmid + 5 U at 37℃.
• Exonuclease Activity: No degradation of 15 ng dsDNA fragments after 16 h with 5 U at 37℃.
• Blue/White Screening: <1% white colonies after ligation and transformation into E. coli XL1-Blue.

Protocols

1) Sticky-End Ligation (20 μl)

• Vector DNA: 20–100 ng
• Insert DNA: 3:1–10:1 molar ratio
• 10× Buffer: 2 μl
• T4 DNA Ligase (Fast): 1 U (0.2 μl)
• Nuclease-Free Water: to 20 μl

Incubate at 22℃ for 10 min; use 1–5 μl for transformation.

2) Blunt-End Ligation (20 μl)

• Vector DNA: 20–100 ng
• Insert DNA: 3:1–10:1 molar ratio
• 10× Buffer: 2 μl
• 50% PEG: 2 μl
• T4 DNA Ligase (Fast): 5 U (1 μl)
• Nuclease-Free Water: to 20 μl

Incubate at 22℃ for 1 h; use 1–5 μl for transformation.

3) Linear DNA Circularization (50 μl)

• Linear DNA: 10–50 ng
• 10× Buffer: 5 μl
• T4 DNA Ligase (Fast): 5 U (1 μl)
• Nuclease-Free Water: to 50 μl

Incubate at 22℃ for 10 min; use 1–5 μl for transformation.

4) Adapter Ligation (20 μl)

• Linear DNA: 100–500 ng
• Phosphorylated adapters: 1–2 μg
• 10× Buffer: 2 μl
• 50% PEG: 2 μl
• T4 DNA Ligase (Fast): 2 U (0.4 μl)
• Nuclease-Free Water: to 20 μl

Incubate 22℃ for 10 min → Heat inactivate at 65℃ 10 min or 70℃ 5 min. Can also be used with CutOne™ buffer + ATP (1 mM) for streamlined ligation-digestion workflows.

Notice

• Strongly inhibited by NaCl or KCl >200 mM.
• Do not exceed 10% ligation mixture volume in competent cells.
• Ligation products may show band shifting/smearing on gels; heat-inactivate or purify if needed.
• PEG enhances blunt-end ligation (recommended 5% w/v PEG 8000).
• Transformation efficiency increases with longer ligation (up to 1 h).