This prevents vector self-ligation because the enzyme ligase requires both a 5′ phosphate and a 3′ OH to join the two ends in recircularization of the vector (see Ligation). During dephosphorylation, the enzyme alkaline phosphatase removes the 5′ phosphate groups at the ends. The MCS, if available, is often the first choice for insertion, as the region is specifically designed for cloning.Īfter restriction digestion, dephosphorylation of the vector may be necessary to prevent self-ligation, especially if the resulting ends of vector digestion are compatible or blunt. Vector restriction sites can be found on the vector map, or can be mapped using free online tools such as RestrictionMapper. The choice of restriction enzymes depends upon the presence and location of their recognition sequences on the vector and the insert, and their compatibility for ligation. The first step in preparing the vector for traditional cloning is to create an insertion site by restriction digestion. For instance, the pUC18 vector expresses the lacZα gene encoding the alpha peptide of beta-galactosidase which, in combination with X-gal, allows color selection of bacterial colonies formed after cloning (learn more about blue/white selection in colony screening). In some vectors, the MCS is located within a gene that serves as a marker and permits screening for clones into which the insert has been spliced successfully. All cloning vectors based on plasmids contain a number of crucial elements, including a bacterial origin of replication to efficiently propagate within the bacterial host cell single restriction enzyme site(s) or, more commonly, a multiple cloning site (MCS) that contains a number of restriction enzyme sites to allow ready addition of an insert of interest and a marker (e.g., antibiotic resistance) to select for bacteria after successful uptake of the vector. Efficient - blunting and phosphorylation of 0.Vectors used in traditional cloning methods are based on plasmids, which are double-stranded, circular DNAs that replicate inside bacteria independently of the genomic DNA.T4 PNK adds 5'-phosphates to ends of unphosphorylated DNA fragments, such as PCR products. The 5'→3' polymerase activity of the End Repair Enzyme Mix fills-in 5' protruded DNA ends while 3'→5' exonuclease activity removes 3'-overhangs. Samples such as fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated), restriction enzyme digested plasmid DNA, double stranded cDNA, and PCR products containing dA overhangs are all compatible with the kit.ĭuring the DNA end repair reaction, fragmented DNA is converted into blunt-end DNA containing a 5'-phosphate and 3'-hydroxyl groups. The 10X End Repair Reaction Mix contains an optimized reaction buffer, ATP, and dNTPs. The End Repair Enzyme Mix contains an optimized mixture of T4 DNA Polymerase and Klenow Fragment to achieve highly effective blunting of fragmented DNA, and T4 Polynucleotide Kinase (PNK) for efficient phosphorylation of DNA ends. Thermo Scientific Fast DNA End Repair Kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.Īll components of the kit contain premixed reagents to reduce pipetting steps and provide convenience.
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