Restriction enzymes transformation

Each of the -6000 PCR products was then co-transformed into yeast along with the recipient vector that had been linearized using a restriction enzyme that digests the plasmid at the desired cloning site. The 70 bp of homologous flanking sequence on each end of the PCR products is sufficient for the yeast homologous recombination system to act upon and insert the PCR product into the vector (Hudson et al., 1997 Ma et al., 1987). 

Co-transform with unrelated Verify interaction by yeast mating Diagnostic restriction enzyme... 

Some bacteria are able to take up DNA from their surroundings and exchange regions with their own DNA. This recombination is the basis of a primitive sexual process known as transformation. However, recombination with DNA from species other than the same as the bacterium is unlikely to be beneficial and it is likely that restriction enzymes have evolved to destroy such foreign DNA. 

An extension to this existing kit is the QuikChange Multi, which enables multiple mutations at different sites. It uses only one mutated oligonucleotide primer, in contrast to the kit described above, but follows the same procedure using the restriction enzyme Dpnl. The single-stranded plasmid is converted into a double strand in vivo after transformation into an E. coli host. 

After transformation, analyze the transformant with colony PCR or plasmid mini-prep procedures and check the restriction enzyme sites by digestion. 

The cloning of functional genes from natural microbial consortia is dependent on the high quality of the extracted and purified environmental DNA since the enzymatic modifications required during the cloning steps are sensitive to contamination by various biotic and abiotic components that are present in environmental ecosystems. For example, extraction of DNA from soils always results in coextraction of humic substances, which interfere with restriction enzyme digestion and PCR amplification and reduce transformation efficiency and DNA hybridization specificity [19 -22], Therefore, extraction methods have been developed to remove or minimize contamination of the purified DNA by humic or other interfering substances. Several protocols for the isolation of bacterial community DNA from various environmental samples have been reported in recent years. These methods are based either on re-... 

If possible, digest this product using a restriction enzyme (E in Rg. 2) that has had its site deleted by the PCR reaction. This should decrease the number of background parental construct colonies obtained. Transform E.cob and plate on a selective medium. 

Figure 7 Cloning by restriction digest and ligation. Both the PCR-amplified gene of interest and the target vector are digested with the same restriction enzymes (or with enzymes that produce compatible ends). The short section of overhanging sequence (called sticky ends ) are complementary to each other. The digested DNAs are mixed, treated with ligase, and transformed into a suitable Escherichia coli host strain to produce a new recombinant DNA molecule with the gene of interest specifically inserted into a host vector.

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