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Messaggio inserito da imy85x

no aspè....in pratica...prendi PGEM ...dove inserisci una certa sequenza di tuo interesse,poichè c'è un promotore forte in PGEM,lo linearizzi,gli dai la polimerasi specifica per quel promotore e i precursori dell'rna(di cui qualcuno marcato).L'enzima si lega al promotore e trascrive questo pezzo...quando arriva alla fine del pezzo la trascrizione si ferma perchè non c'è più stampo...(per questo motivo si dice RUN-OFF...perchè la trascrizione termina)ed hai quindi la sonda ad rna...(forse così si capisce meglio)

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There is a scientific difference which is not semantic!! Run-on transcription refers to in vitro nuclear
transcription analysis where preinitiated complexes in intact nuclei are permitted to elongate the
nascent RNA transcript 200-400 bases after addition of XTPs. Run-off transcription refers to
in vitro transcription where a naked, linearized DNA template is mixed with polymerase and other
goodies and it initiates, elongates then falls off the end of the linear template. Hence run-OFF.

I teach this in Gene Expression for beginners and every year I fine students 2 points for making this
sloppy mistake.


Ken Krauter
Department of Cell Biology
Albert Einstein College of Medicine
1300 Morris Park Ave.
Bronx, NY 10461
Tel. (718) 430-3509
FAX. (718) 829-7619
Email ksk at ksksun.ca.aecom.yu.edu

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Run-off transcription
Characterization of trans-acting factors

This assay is based on the observation that the transcription complex will fall off from a DNA template if complex reaches the end of the template. A restriction enzyme site within the gene can therefore be chosen generate run-off transcripts of a proper length. As long as the RNA is initiated at the same place, the run-off transcripts should have an uniform length . This method eliminates the complication from the transcription elongation. The product can be assayed by direct labeling the RNA product during transcription or by other methods that can quantitate the unlabeled RNA from trasncription reaction, such as the primer extension or RNase protection assays.

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Messaggio inserito da viodice88

dopo anni sono capitata qui e..anche io ho l'esame con la fucci, aiutoooo!! vorrei chiedere..non è che qualcuno di voi magari ricorda cos'è un run-on? e soprattutto come si fa??

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