Seamless Cloning Assay kit

1. Preparation of linearized vector Two choices: (1) Select the appropriate site on the target vector plasmid, perform single or double digestion, and then purify after digestion; (2) Reverse PCR amplifies the vector, and designs a pair of complementary primers at the linearization site for amplification. Note: Single digestion of vector plasmid can easily lead to incomplete cleavage and self-linking , resulting in false positives, so double digestion should be selected as much as possible. Be sure to tap the gel to recover the linearized vector, otherwise the residual circular plasmid will cause background colonies. Generally, the linearized vector concentration after recovery needs to be> 15ng / µl. 2. Preparation of target gene fragments Primer design: (1) PCR primer must contain two parts: 5 'end of the primer must contain 15-25 bp which are completely homologous to the end of the vector. 3' end of the primer must contain the target gene specific primer sequence. (2) 3 'end of each primer must be specific for the target gene, with a length of 15-25 bp, GC% of 40% -60%, and an annealing temperature Tm of 58 - 65 ° C. PCR product is run through the gel and purified by tapping. (3) When multiple fragments are inserted, 5 'end of the first and last fragment primers must contain 15-25 bp bases that are completely homologous to the end of the vector. 5 'end of the remaining intermediate primers must contain 15 bp homologous sequence with the previous fragment, and 3' end must contain 15 bp homologous sequence with the latter fragment. 3. Refer to the following table to prepare the reaction system on ice: Reaction conditions: insert single fragment generally requires 30 min reaction at 50 ° C; with the increase of the fragment number and the increase of length, the reaction time is extended, generally 45-60min. Note: The amount of linearized vector and the target fragment can be adjusted according to their respective concentrations and the length of the fragment. The molar ratio of the target fragment to the linearized vector is about 3: 1, too high or too low will reduce the recombination efficiency. 4.Transformation and identification of positive clones (1) Take 100μl of competent cells to be transformed and thaw them on ice. (2) Add 10μl of the reconstituted reaction solution (as per step 3) to the above competent cell suspension, mix gently without shaking, and place the mixture on ice for 30 min. (3) Heat shock in a water bath at 42 ° C for 90 sec, immediately store on ice for 2 min, then add 500μl of LB medium (without resistance), and shake at 200rpm in a shaker at 37 ° C for about 1 h. (4) Take 200-300μl of the above transformation mixture and spread it on selective solid medium (containing the corresponding antibiotics). (5)Pick the clones on the plate for colony PCR, or double-digest the plasmid to identify positive clones.

Seamless cloning is a fast and efficient Gibson Assembly DNA directional cloning technology, which can simultaneously clone multiple DNA fragments into any vector, and the final constructed clone without any additional base sequence, so called "Seamless cloning". Compared with the traditional gene cloning method, seamless cloning kit has many advantages: (1) simple operation and only needs one reaction; (2) there is no requirement for the enzyme digestion site, the target fragment can insert into any position of any vector; (3) No extra sequence will be introduced between the connected fragments; (4) The positive cloning rate is high, which reduces the workload of cloning screening. The kit only needs to linearize the vector at the insertion site, at the same time, mix the linearized cloning vector and the homologous sequence PCR fragment (15-25bp primers at both ends of the target fragment and the vector insertion site) with appropriate ratio, and 2 × Master Mix is added. Under the catalysis of the recombinase, the directional cloning could be completed quickly at 50 ℃ within 30 minutes, and the positive rate is as high as 90%. 2 × Master Mix in the kit is pre-mixed with DNA exonuclease, DNA polymerase, ligase and recombination buffer, and added special enzyme activation components, which can significantly improve the efficiency of recombination cloning, can achieve more than one sequence cloning of up to 3-6 fragments.

Compared with the traditional gene cloning method, seamless cloning kit has many advantages: (1) simple operation and only needs one reaction; (2) there is no requirement for the enzyme digestion site, the target fragment can insert into any position of any vector; (3) No extra sequence will be introduced between the connected fragments; (4) The positive cloning rate is high, which reduces the workload of cloning screening.

Store at -20℃ for 12 months, avoid freeze-thaw cycle cycle.

Shipping with dry ice.

For research use only.

Guarantee and Disclaimer:

After receiving the product, if finds that the product is mismatched, damaged or missing components, please keep the original package and submit the objection to the company by mail within seven working days. Failure to file an objection within the time limit is considered qualified.

When the buyer keeps the product, it should be kept in accordance with the storage conditions shown in the product label and the manual. If the product quality is caused by improper storage, it will not be guaranteed.

When the buyer tests the product, they should be submitted by the beginning of use when found the quality issue, rather than when the product was used or used up, in order to prepare our products for recycling and confirm the quality of the products. If it is the quality problem, our company is responsible for exchange or return. In the event of a claim, our company will compensate the discretion within the scope of the product price limit and will not accept any part of the value of the product itself. Since the date of receipt, the product has not been reflected for more than three months and should not be returned.

The products provided by our company are for research use only and should not be used for clinical diagnosis or treatment. If a unit or individual changes the use of our products without authorization, we will not bear any responsibility.