Restriction Enzymes at NEB: Over 30 years of Innovation.
When selecting a restriction site(s) to add to the primers, it is important to determine which site(s) will be compatible with your selected vector, whether directional cloning is desired and, most importantly, confirm that the recognition site(s) does not occur within the gene or DNA fragment. These additional bases provide sufficient DNA for the restriction enzyme to bind the recognition site and cut efficiently. When adding restriction sites to a PCR primer, it is recommended to include 6 bases between the recognition site and the 5’ end of the primer. In order to generate compatible ends, it is common to add restriction sites to the 5’ end of both PCR primers. The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any source of DNA, to be cloned. In this case, it is essential that the gene be inserted in the correct orientation and in frame with the transcription promoter. The gene of interest is most commonly subcloned into an expression vector for improved protein expression and/or addition of a purification tag. Restriction enzymes that have a recognition site within the multiple cloning site (MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments. Subcloning requires the use of 1-2 restriction enzymes that cut immediately outside the insert fragment without cutting within the insert itself. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected vector DNA.
SERIAL CLONER COHESIVE ENDS SERIAL
Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8 consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in larger DNA fragments. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid. Restriction enzymes can also be used to generate compatible ends on PCR products. The DNA to be cloned can vary widely, from genomic DNA extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector to another (subcloning). Overall, the Serial Cloner app provides you with a useful set of tools for DNA cloning and sequence visualization.Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. By default the Help window is permanently displayed and provides you with tips for the window or field hovered by the mouse cursor.
The interface is intuitive and provides quick access to all the important features.Īlthough the program is clearly designed for users accustomed with molecular biology analysis, it features an extensive documentation that helps beginners get used to the features. This enables you to work with sequences created by other laboratories in order to compare the results.ĭuring our tests Serial Cloner required insignificant resources and had no impact on the other applications even when generating complex graphical maps. The application allows you to import the data from multiple programs and supports various file formats such as Vector NTI, MacVector, ApE, DNAstar, pDRAW32 and GenBank. You can also export the collection of features, align protein sequences and reverse translate proteins with minimum effort. If the analysis results are required in another document you can simply copy-paste the map or save it as an image on your hard drive. The graphical map supports further formatting which enables you to specify the displayed elements. The program can help you find restriction sites and build a text restriction map which enables you to add multi-frame translations. It is designed to handle all the data in a single interface which makes it easy to use when manipulating sequences. You can use this tool for analyzing the datasets, selecting certain fragments and calculate Tm values with just a few clicks. It deals with the analysis and visualization of sequences in the field of molecular biology. Serial Cloner is a powerful tool for the laboratory staff and the students who work with nucleotide and peptide sequences.
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