Product descriptionTOPspeed™ DNA Ligation Kit is formulated for quick ligation of DNA fragments with cohesive ends within 5 min at room temperature. This product uses T4 DNA Ligase (Cat.# M001) and 2X TOPspeed™ DNA Ligation buffer.Characteristics- 5-min ligation reaction with all types of DNA fragment- Reaction at room temperature- Rapid and efficient TOPspeed™ Ligation bufferApplications- General cloning- Blunt-end cloning- TA cloning- Library construction
- Linker ligation
- Prewarm 2X TOPspeed™ DNA Ligation buffer to room temperature prior to use. We recommend that at least 20 μl are used per reaction. Avoid frequent cycles of freezing and thawing of the buffer.
- Calculate the mole number of vector and insert DNA by measuring their concentration prior to ligation. Use 2 to 3 times (cohesive end DNA ligation) or 5 to 6 times (blunt end ligation) more insert DNA than vector DNA.
- Transformation efficiency reduces rapidly if TOPspeed™ DNA Ligation mixture is heated.
- Incubation longer than 1 hr is likely to reduce transformation efficiency. Therefore, we recommend that transformation should be carried out after 5-30 min of ligation reaction.
Figure 1. Ligation of blunt-end fragments using TOPspeed™ DNA Ligation Kit.
Bacteriophage λ DNA was digested with EcoR V (Cat.# R018) to generate blunt-ended restriction fragments. EcoR V-cleaved λ DNA (500 ng) were ligated by the use of TOPspeed™ DNA Ligation Kit (Cat.# EZ003). Periods of incubation are as indicated (in min). Ligation products were analyzed in a 1% agarose gel. Lane 1, EcoR V-cleaved λ DNA only as a negative control; Mw, 1 kb (+) Ladder Marker (Cat.# DM003).
Figure 2. Enhanced ligation efficiency with TOPspeed™ DNA Ligation kit.
Vector DNAs were cleaved with either Hind III (Cat.# R008), EcoR V (Cat.# R018), or Hind III/Xba I (Cat.# R013). Vectors digested with Hind III or EcoR V were dephosphorylated prior to ligation reaction. The vector DNAs prepared this way were mixed with insert DNAs precleaved with the corresponding restriction enzyme(s). Ligation reactions were carried out with T4 DNA Ligase (Cat.# M001) at 16℃ for 16 hr (green box, standard) or with TOPspeed™ Ligation Kit (Cat.# EZ003) at 25℃ for 5 min (red box, TOPspeed). Ligation products were directly transformed into chemically competent E. coli DH5 (Cat.# CP010) to measure ligation efficiency. Fold increases in the numbers of transformants were presented; the number of transformants obtained from standard ligation procedure was assumed to be 1.