The critical concentration (Cc) is the concentration of free G-actin at which the filaments are formed.

TLS Online TPP Program

#Id: 2641

#Unit 13. Methods in Biology

The size of this run-off transcript locates the transcription start site, and the amount of this transcript reflects the efficiency of transcription. 

TLS Online TPP Program

#Id: 2642

#Unit 13. Methods in Biology

Notice that S1 mapping and primer extension are well suited to mapping transcripts made in vivo; by contrast, run-off transcription relies on transcription in vitro.

TLS Online TPP Program

#Id: 2643

#Unit 13. Methods in Biology

After you have identified the physiological transcription start site by S1 mapping or primer extension you can use run-off transcription in vitro.

TLS Online TPP Program

#Id: 2644

#Unit 13. Methods in Biology

A variation on the run-off theme of quantifying accurate transcription in vitro is the G-less cassette assay.

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#Id: 2645

#Unit 13. Methods in Biology

In G-less cassette transcription, a promoter is fused to a double stranded DNA cassette lacking G’s in the nontemplate strand, then the construct is transcribed in vitro in the absence of GTP. 

TLS Online TPP Program

#Id: 2646

#Unit 13. Methods in Biology

A G-less cassette, or stretch of nucleotides lacking guanines in the nontemplate strand, is inserted just downstream of the promoter. This template is transcribed in vitro with CTP, ATP, and UTP, one of which is labeled, but no GTP.