Gene Cloning-Single Fragment Seamless Cloning Kit
Gene Cloning-Single Fragment Seamless Cloning Kit

Gene Cloning-Single Fragment Seamless Cloning Kit

This is a product developed based on seamless cloning technology. As a new generation cloning method, it does not rely on tedious enzyme digestion, connection steps, nor does it require end filling operations. Based on the recombination of DNA fragments with the homologous sequence of 15-25 nt at the end of the linearized vector, the inserted fragments can be cloned to any location on any linear vector.


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Article No.Product NameChinese NameSpecificationsPrice（Yuan）
DC00201Single Fragment Seamless Cloning Kit单片段无缝克隆试剂盒25 rxns688

This is a product developed based on seamless cloning technology. As a new generation cloning method, it does not rely on tedious enzyme digestion, connection steps, nor does it require end filling operations. Based on the recombination of DNA fragments with the homologous sequence of 15-25 nt at the end of the linearized vector, the inserted fragments can be cloned to any location on any linear vector. The vector has a very low background of self connection, making it a simple, fast, and efficient DNA directed cloning technology



The single fragment seamless cloning kit can complete single fragment recombination in as little as 5 minutes, and the positive rate is above 95%. The auxiliary factors added in Mix effectively improve the cloning positive rate; The optimized reaction system can tolerate impurities contained in unpurified PCR products to a certain extent. The upgraded seamless cloning kit has a higher positive rate and better compatibility.



Descriptions

Causes

Solution

Low transformation efficiency.



Low efficiency of competence cells.

Use newly prepared or properly preserved competence cells.

Poor proportion of DNA fragment.

Prepare the reaction system according to the most suitable amount and proportion recommended in the instruction manual. Concentration determination of carrier and inserted fragment: If the linearized vector and inserted fragment have been purified and the electrophoresis detection band is single or without Smear residue, concentration determination can be carried out using instruments based on spectrophotometry such as ultra micro nucleic acid protein detector. However, the concentration value is reliable only when A260/A280 is between 1.8 and 2.0; If the linearized vector and inserted fragment have not been purified, agarose electrophoresis can also be used to determine sample concentration.

Poor purity of DNA fragments.

Perform gel extraction and purification of the vector and inserted fragments. Due to the inhibitory effect of metal ion chelating agents such as EDTA on seamless cloning reactions, the purified product should be dissolved in ddH2O and buffer solutions such as Tris- EDTA should not be used.

Excessive reaction products.

In the transformation system, the volume of seamless cloning reaction products should not exceed 10% of the volume of competence cells.

Large number of clones without inserted fragments.

Incomplete vector linearization.

When preparing linear vector by digestion, increase the use of rapid endonuclease and prolong the reaction time, and purify the digestion products by gel extraction.

Contaminated by plasmids with the same resistance.

When using plasmid as template for PCR amplification of inserted fragments, use pre linearized plasmid as amplification template, use methylation sensitive Endonuclease such as DpnI to treat the amplified products, or purify the products by gel extraction.

Insufficient antibiotics in the culture medium.

Ensure the use of the correct antibiotics and use freshly prepared solid culture media containing antibiotics.

Large number of clones with wrong fragments.

Non-specific amplification products.


Optimize the PCR system, improve amplification specificity, or purify amplification primers for overlapping sequences of PCR products by gel extraction.



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