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The Thermo Scientific Rapid DNA Ligation Kit enables fast sticky-end or blunt-end DNA ligation in only 5 minutes at room temperature (see Figure 1 in Supportig Data).
The kit contains T4 DNA ligase and a specially-formulated 5X rapid ligation buffer optimized for fast and efficient DNA ligation. Fast ligation efficiency is equal to that obtained with T4 DNA ligase in a standard 1 hour ligation. The ligation reaction mixture can be used directly for bacterial transformation with the TransformAid Bacterial Transformation Kit or with other conventional transformation procedures.
Prior to electroporation, it is necessary to inactivate T4 DNA ligase (see Reference 1) by chloroform extraction or spin column purification, e.g. with Thermo Scientific GeneJET PCR Purification Kit (K0701).
Ligation of sticky- and blunt-end DNA fragments with the Rapid DNA Ligation Kit. Blunt-end ligation. pUC19 DNA/SmaI dephosphorylated vector was ligated with Lambda DNA/PvuII fragment (2.3 kb). Sticky-end ligation. pUC19 DNA/PstI dephosphorylated vector was ligated with Lambda DNA/PstI fragment (2.1 kb). The efficiency of ligation was determined by transformation of E. coli XL1-Blue cells.