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Finding an Individual Clone in a DNA Library

How do we find the piece of DNA we want in a library Finding an Individual Clone in a DNA Library [Pg.384]

The nitrocellulose disc is treated with a denaturing agent to unwind aU the DNA on it. [The DNA has become accessible by disruption (lysis) of the bacterial cells or phage.] After it is denatured, the DNA is permanendy fixed to the disc by treatment with heat or ultraviolet light. The next step is to expose the disc to a solution that contains a single-stranded DNA (or RNA) probe that has a sequence complementary to one of the strands in the clone of interest [Pg.384]

Master plate of bacteria colonies (or phage plaques) [Pg.385]

If the nucleotide sequence of the desired DNA segment is not known and no probe is available, a complication arises. If the gene of interest directs the synthesis of a given protein, one chooses a vector that allows cloned genes to be transcribed and translated. If the presence of the desired protein can be detected by its function, that serves as the basis for detecting it. Alternatively, labeled antibodies can be used as a basis for protein detection. [Pg.385]

The genome is digested with restriction enzymes and the pieces are cloned into vectors and transformed into cell lines. [Pg.385]




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