Efficient DNA Subcloning through Selective Restriction Endonuclease Digestion
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چکیده
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Efficient DNA subcloning through selective restriction endonuclease digestion.
Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E. coli-based plasmids. The method increases yield while decreasing laboratory resource and time utilization. By using donor and acceptor sequences that contain unique restriction sites found only outside of...
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The polymerase chain reaction (PCR) has become the most widely used technique for the manipulation of DNA with applications for cDNA cloning, gene cloning, polymorphism detection, mutagenesis and allele-specific diagnosis (8). All of those applications have been extended significantly by the recent development of “long” PCR, a technique that makes it possible to amplify DNA fragments up to 40 k...
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Present-day DNA technology is partially dependent upon the ability of investigators to cut DNA molecules at specific sites with enzymes called restriction endonucleases or restriction enzymes. The most useful of these enzymes, which occur naturally within bacteria, recognize particular "target" or recognition sequences (restriction sites) within a DNA double helix, and catalyze the cleavage of ...
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A difficulty that is encountered when attempting to insert a PCR-amplified product or DNA fragment of interest into a particular vector is the presence within the insert of one or more internal restriction endonuclease (RE) sites identical to those selected for the flanks of the insert. Our method circumvents this problem by partially protecting internal RE sites while flanking sites for the sa...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 2000
ISSN: 0736-6205,1940-9818
DOI: 10.2144/00284st01