The Retinoblastoma Protein by Pedro G. Santiago-Cardona

Author:Pedro G. Santiago-Cardona
Language: eng
Format: epub, pdf
Publisher: Springer New York, New York, NY

BACs

E. coli

Circ. plasmid

Up to 300

PACs

E. coli

Circ. plasmid

100/300

YACs

Saccharomyces cerevisiae

Linear chromosomes

100/2000

Abbreviations: BACs bacterial artificial chromosomes, PACs P1 derived chromosomes, YACs yeast artificial chromosomes, E. coli Escherichia coli, Circ. plasmid circular plasmid [20]

After harvesting and lysing the host cells, the cloned DNA is purified from the host chromosomal DNA and cellular material using commercial kits (Qiagene, Thermo Fisher Scientific, Invitrogen). Bacterial cells containing the clone of interest are usually grown in media that selects for the clone by use of an antibiotic, or in the case of yeast, in media which lacks a particular nutrient. Detailed description for growing bacteria and for isolating DNA are described by Garimberti E and Tosi S [20]. In order to label the DNA fluorescently, the most frequently used protocol is nick translation (see Note 6 ). The procedure allows the incorporation of fluorescently labelled dUTPs. A wide range of fluorescent dyes are available for labeling. The most frequently used are: fluorescein isothiocyanate (FITC) and different cyanine fluorophores . A wide range of fluorescently labeled nucleotides are available from different companies (e.g., Thermo Fisher Scientific, MoBiTec, and PromoKine). The easiest way to perform nick translation is to use commercial kits, such as the ones available from vendors such as Enzo, GIBCO, Abbott Molecular, and Roche Ltd. Below is the modification of a nick translation protocol for plasmid or cosmid probes (adapted from Fred Waldman’s Laboratory UCSF).

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