DNA ligases catalyze the formation of phosphodiester bonds at single-stranded DNA breaks in double-stranded DNA in vivo, and T4 DNA Ligase is a type of DNA ligase. This kit contains Fast Ligase and the optimized Fast Ligation Reaction Buffer (2X) which can be used as a simple system for rapid DNA ligation reactions. The ligation of sticky-end or flat-end DNA insert fragments to plasmid vectors can be efficiently accomplished within 5-10 min at room temperature (25℃), and the ligation efficiency of Fast Ligation is equivalent to that of conventional ligation with T4 DNA Ligase for 1 hour. The ligated product can be directly used in subsequent transformation experiments without purification.
1. Prepare the following mixture in a Nuclease-Free centrifuge tube:
Component | Volume |
Fast Ligation Reaction Buffer (2X) | 10 μl |
Vector DNA | X μl |
Insert DNA | Y μl |
Nuclease-Free dH2O | Up to 20 μl |
Fast Ligase | 1 μl |
2. Calculation of Molarity of Ends:
Molarity = [(µg/µl) ÷ (base pairs x 650 daltons)] x 2 ends
3. Blow the mixture gently to mix well and centrifuge instantaneously.
4. Leave the mixture at room temperature (25°C) for 5-10min.
5. Transfer 1-5μl of the reaction solution into 50μl of receptor cells for subsequent experiments, or store at -20℃.
1. Fast Ligase is added last.
2. Fast Ligation Buffer should be thawed in advance at room temperature and mixed well by shaking up and down.
3. The optimal molar ratio of vector to inserted DNA is 1:3.
4. In the case of vector single digestion for insertion of exogenous fragments, care should be taken to dephosphorylate the vector to avoid plasmid self-association.
5. The enzyme product should be kept on ice during the experimental operation and stored at -20°C immediately after the experiment.
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