Golden Gate Assembly Kit for BsmBI-Based Cloning

Golden Gate Assembly Kit for BsmBI-based cloning enables one-step, seamless DNA assembly using Type IIS restriction enzyme digestion and T4 DNA ligase-mediated joining of fragments with designed complementary overhangs.

This Golden Gate Assembly Kit shares the same single-step precision cloning strategy, popularly known as “Golden Gate Assembly”, that relies on the unique properties of type IIs restriction enzymes to generate compatible ends on DNA fragments that are then joined together by the T4 DNA ligase. The Kit is particularly adept at assembling difficult to-clone sequences such as repetitive and very small sequences (＜100 bp), gene variants, and TAL (transcription activator-like) effector genes.
Type IIS restriction enzymes bind to their recognition sites but cut the DNA downstream from that site at a positional, not sequence specific, cut site. Thus, a single Type IIS restriction enzyme can be used to generate DNA fragments with unique overhangs.
The cloning procedure is as follows: Design recognition sites for type IIS restriction enzymes outside the cleavage sites of the target gene. Assembly of digested fragments proceeds through annealing of complementary four base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, so no further cutting is possible. The vector contains sticky ends complementary to those of the target gene, enabling ligation without introducing extraneous sequences, thereby achieving seamless cloning.
The Golden Gate Assembly Kit (BsmBI), based on the above principle, contains all enzymes required for restriction digestion and ligation in a ready-to-use Mix for convenient pipetting. It supports ligation of up to 16 fragments in a single reaction, fully satisfying diverse experimental requirements.

• Product: Golden Gate Assembly Kit (BsmBI)
• REF No.: EG25207V and EG25207S
• Sizes: 10 reactions and 50 reactions
• Components:
  • Golden Gate Mix (BsmBI)
  • 10× T4 DNA Ligase Buffer
• Component volumes:
  • EG25207V: 10 μl Golden Gate Mix (BsmBI), 1 ml 10× T4 DNA Ligase Buffer
  • EG25207S: 50 μl Golden Gate Mix (BsmBI), 1 ml 10× T4 DNA Ligase Buffer
• Cloning principle: Type IIS restriction enzyme digestion plus T4 DNA ligase-mediated assembly
• Application: seamless cloning
• Assembly capacity: up to 16 fragments in a single reaction
• Supplier-highlighted difficult templates/applications:
  • repetitive sequences
  • very small sequences below 100 bp
  • gene variants
  • TAL effector genes
• Storage: -20°C
• Shelf life: 2 years