#Gene construction kit free download Activator
Using this insert restriction enzyme cutting-free cloning (ICFC) method, we successfully constructed a pSG5- STAT3 plasmid expressing the signal transducer and activator of transcription 3 ( STAT3) gene.
Here, we introduced a novel method to generate inserts with desired cohesive ends by denaturing and re-annealing two PCR products. However, they either rely on special enzymes or are too complex. These methods are effective and reliable. To date, there were several reports of restriction endonuclease digestion-independent cloning methods, including RSFC (restriction site free cloning), SLiCE (seamless ligation cloning extract), SLIC (sequence and ligation-independent cloning), Gibson assembly, CPEC (circular polymerase extension cloning). All these drawbacks result from the requirement of digesting the inserts to generate cohesive ends. However, this is a time- and cost-consuming procedure. The T-A cloning technology can overcome this disadvantage. Secondly, the low efficiency of digesting the terminal ends of PCR (polymerase chain reaction) products by restriction enzymes often leads to failure of the plasmid construction procedure. Therefore, the restriction enzyme cutting sites within the inserts should be avoided, which sometimes leads to failure in selecting suitable restriction enzymes, especially when the aim is to transfer the same inserts into different vectors. Firstly, this method relies on restriction enzyme(s)-cutting of the inserts. However, there are several disadvantages. The traditional restriction enzyme digestion and re-ligation method is a routine way to construct plasmids. This also depends on the use of plasmids. In addition, site-directed mutagenesis and gene fragmentation are essential in exploring the role of single amino acids or domains in proteins. Plasmids are also used as carriers of reporter genes to analyze the biological activities of corresponding transcription factors or explore the molecular mechanisms underlying the expression regulation of target genes. Construction of fusion proteins also allows detecting, localizing and purifying the proteins, as well as investigating the protein–protein interactions more easily. What is more, plasmids are one of the simplest but most efficient tools to explore the biological roles of genes/proteins, either by gene overexpression or gene silencing using RNA interference. They have been widely used in the production of many biologically active agents, such as biotechnological enzymes, medical drugs (such as recombinant interferons, insulins), healthcare products and even foods. Plasmid-mediated target gene amplification and over-expression are routine strategies in genetic engineering, biological investigation and industrial production. It made the selection of restriction endonucleases and vectors easier, as well as simplified and shortened the cloning process. The method proved to be simple and efficient. Using this method, we successfully constructed a STAT3 (signal transducer and activator of transcription 3) expression plasmid. Finally, they were ligated to the restriction-endonuclease digested vectors. Inserts with desired cohesive ends were generated by successively mixing, denaturing and re-annealing these DNA fragments. Two kinds of DNA fragments containing the same gene but different terminal sequences were amplified by PCR. To address these problems, we employed an insert cutting-free cloning (ICFC) method in this work. It is time-consuming sometimes there are no suitable restrictive digestion sites and the efficiency of digestion of the polymerase chain reaction (PCR) products is low. However, there are some drawbacks of this method due to its nature. They are often constructed by the traditional restriction digestion/ligation cloning method. Plasmids are important tools for producing biological reagents and performing molecular biological investigations.
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