High Efficiency DNA Ligation Using the Fast-Link™ DNA Ligation and Screening Kit

T4 DNA ligase reactions are widely used in molecular biology for subcloning, ligation-mediated PCR (LM-PCR) and DNA library construction. Conventional ligation requires a long incubation period (typically overnight) at temperatures ranging from 12-22°C. Using Epicentre's Fast-Link™ DNA Ligation and Screening Kit,* we report that ligation reactions containing blunt-end DNA or PCR products can be performed in 15 minutes at room temperature with higher efficiency than conventional ligation. This system would be useful for applications where high-efficiency DNA ligation is required.

Methods and Results

Comparison of Fast-Link rapid ligation and conventional ligation with blunt-end, double-stranded DNA

To compare Fast-Link ligation with conventional ligation, 10 µg of pBluescript® SK (Stratagene) were digested with EcoR V to generate blunt-ended, linearized plasmid that was then treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim) to prevent self-ligation. Blunt-ended PCR fragments were generated using Vent® DNA polymerase (New England Biolabs) with 30 cycles of amplification. The amplified fragment (350 bp) was subjected to gel electrophoresis in a 1.2% agarose gel and the gel fragment containing the amplified DNA was excised. Extraction of the DNA was performed using the Geneclean® kit (BIO 101) according to the manufacturer's instructions. For conventional ligation, 50 ng of the insert were incubated with 150 ng EcoR V-treated plasmid, 4U of T4 DNA ligase purchased from Stratagene, standard buffer and 1 mM ATP in a total volume of 15 µl. The reaction was incubated at 22°C for 12 hours. Ligation using the Fast-Link Kit was performed according to the manufacturer's instructions. The ligation reaction contained 50 ng of insert DNA, 150 ng of digested plasmid, 2U of Fast-Link T4 DNA Ligase, and 0.5 mM ATP in a total volume of 15 µl in 1X Fast-Link Ligation Buffer. The reaction was incubated for 15 minutes at room temperature, and then at 70°C for 15 minutes. 2.5 µl of the ligation mixture from each reaction were used to transform XL2-Blue competent cells (Stratagene), and one-third of the total transformed cells were plated on LB Amp/IPTG/X-gal plates.

Table 1 shows the ligation efficiency calculated from the colony number obtained after a 12-hour incubation of the plates at 37°C. Ligation with the Fast-Link Kit was almost 10-fold more efficient than conventional ligation.