Identifying transcribed sequences in arrayed bacteriophage or cosmid libraries.
This unit describes methods for identifying bacteriophage or cosmid clones that contain sequences present in the mRNA of a cell line or tissue. In the , a radiolabeled cDNA probe is synthesized by reverse transcription of poly(A)+ RNA isolated from the cell line or tissue of interest. The probe is incubated with DNA-cellulose to remove highly repetitive sequences before it is used for hybridization analysis of filters from a phage or cosmid genomic library. After identification of positive clones from the library screen, the same probe can be used to screen Southern blots of restriction enzyme-digested DNA from the positive clones. Support protocols describe preparation and testing of DNA-cellulose.
Hochgeschwender
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National Institute of Mental Health, Bethesda, Maryland, USA.
Brennan
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MB
D001435:Bacteriophages / Q000235:genetics; D002482:Cellulose / Q000031:analogs & derivatives; D003360:Cosmids; D004247:DNA; D018076:DNA, Complementary; D005821:Genetic Techniques; D005826:Genetics, Medical; D015698:Genomic Library; D006801:Humans; D014158:Transcription, Genetic
D018076:DNA, Complementary; C033190:DNA-cellulose; D002482:Cellulose; D004247:DNA