OK, Clon. DNA Ligation Kit

Item No.: AG11802 / AG11803

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Price range: ¥480 through ¥2,200

Product Description

OK Clon The DNA Ligation Kit is an accurate, fast and efficient targeted cloning kit. The kit allows for the fast and efficient targeted and seamless cloning of one or more DNA fragments into any position of any vector in less than 10 minutes without being constrained by the enzyme cleavage sites.OK Clon The exonuclease activity of the 3’-5’ end of the Enzyme cuts the nucleotide from the 3’ end of the linearized DNA strand to form a sticky end, thus the fragment can form a 15-20 bp complementary sequence with the end of the vector, and the fragment can be accurately and efficiently ligated to the vector by annealing. When designing the primer, only the sequence complementary to the 5’ end of the vector is added to the 5’ end of the primer, and the target fragment can be amplified by this primer, and it can be inserted into the vector without the need of restriction enzyme cutting or phosphorylation of the PCR fragment.

Product Advantages

1. Fragments can be inserted into any position of the vector without being restricted by the enzyme cutting site;
2. Rapid reaction, 50 ℃ 10 min can complete the connection reaction;
3. The inserted fragments do not need to be digested and can be directly ligated after PCR amplification by purification.

Product Composition
individual parts making up a compound AG11802 AG11803
5X OK, Clon. Master Mix 20 μl 100 μl
Preservation and transportation

Storage temperature: -20℃ storage
Transportation temperature: dry ice transportation or -20℃ ice bag transportation

Example 1

Use this product with the Linearized Control Vector & Insert (for). OK, Clon.) (Code No. AG11814) in linearized vectors and fragments for cloning experiments:
① Prepare the reaction system according to the table below:

component name experimental group negative control positive control*1
5 X OK, Clon. Master Mix 2 μl 2 μl 2 μl
Linearized Control vector (50 ng / μl) 1 μl 1 μl /
2 kb Positive Control Insert (80 ng / μl) 1 μl / /
RNase free water 6 μl 7 μl Up to 10 μl

*1: Positive control with only 100 pg of circular pUC19 plasmid. No linearized vector and fragments are added.

② Convert all groups to E. coli DH5α Competent cells (Code No. AG11806), cultured bacterial suspension was diluted 2-fold and 100 μl was coated on the plate (LB medium was used in this experiment, with Amp antibiotic, IPTD, and X-Gal added for blue and white spotting screening). The results are shown below, the colonies in the experimental group were uniform and hundreds of monoclonal colonies were obtained.

③ 12 single colonies of the experimental group were picked for colony PCR verification. As can be seen from the electrophoresis results in the figure below, the 12 colonies picked were all positive colonies.

④ The 12 colonies verified as positive by colony PCR were sequenced after plasmid extraction, and the sequences were correct.