Seamless cloning technique from HANBIO is a new, fast and simple cloning method, which can insert one or more target DNA fragments at any site of the plasmid without any restriction enzymes and ligases. Breaking through the traditional double enzyme digestion and linking, the recombinant vector of high efficiency cloning can be obtained by one-step recombinant method, which greatly improves the working efficiency.
The HB-infusion™ seamless cloning kit developed by HANBIO is extremely simple to operate. It only needs to linearize the cloning site of the vector and introduce 15-25 bp homologous sequences that are completely consistent with both ends of the vector cloning site at the 5 'end of the insert PCR primer. The linearized cloning vector and the PCR fragments with homologous sequences were mixed in appropriate proportions and then added into HB-infusion™ Master mix. The directed cloning could be completed quickly by the reaction of DNA exonuclease, DNA polymerase and ligase in the reaction system at 50℃ for 20 minutes. The positive rate was almost 100%.
The Operation is Simple and Fast: The seamless cloning kit from HANBIO does not require any restriction enzymes, ligases, phosphorylation, terminal complementation, etc. The reaction time of the system was only 20 minutes.
Wide Application: Multiple DNA fragments, up to 10, can be linked simultaneously.
Precise and Efficient: No extra sequence is attached, precise directional connection, high positive rate.
The design of primers for gene cloning and the amplification of target DNA fragments are the same as conventional PCR. The only difference is that the end of the vector and the end of the primer should have 15 to 20 homologous bases, and the resulting PCR product will carry 15 to 20 homologous bases with the sequence of the vector.
HB-infusion™ seamless clone principle diagram
| Seamless cloning | Traditional cloning |
| Only one reaction is needed to complete the directed cloning, omitting the digestion, enzyme linking and other processes. | PCR primers are designed by introducing the cleavage site on the vector, and the PCR products are then cloned to the target vector after enzyme cleavage, glue recovery and connection. Or TA connection. |
| There is no requirement for restriction site, and the target fragment can be inserted into any site of any vector. | Need to find suitable enzyme restriction sites, and purchase all kinds of endonuclease enzymes. |
| No other sequences are introduced between the linked fragments. | Low positive rate. |
| Multiple fragments can be cloned simultaneously. | Multiple fragments usually can only be spliced several times. |
Code No. | Component | Usage Counter | Volume |
HB-infusion-20T | 2 x HB-infusion™ Master mix | 20 tests | 200µL |
Positive linearized pUC vector （250 ng） | 5 tests | 25µL | |
Positive control insert (500 ng) | 5 tests | 25µL | |
Storage condition | -20℃ | ||
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