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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: Methods. 2008 Oct 21;47(1):25–36. doi: 10.1016/j.ymeth.2008.10.010

Figure 5.

Figure 5

A gel showing the importance of background subtraction during phosphorimager quantitation. Three promoters (designated N, A, and D) were transcribed under the steady-state reaction conditions with [γ-32P]-ATP labeling. N: N25; A: N25anti; D: DG203. The abortive ladders for each promoter are indicated on the border: N, the far left column; A: the nearby left column; D: the nearby right column. W: the bottom edge of the sample wells. 57: the length of run-off RNA. For quantitation purposes, a no-enzyme control reaction (designated as C) was included. (The control reaction contained N25 promoter, was incubated with enzyme-dilution buffer, and treated through all of the subsequent recovery steps.) One can see the substantial background contribution to the 2- and 3-nt spots. In lane A, N25anti transcription yields slippage products C5, C6, and C7, marked by asterisks. The identity of C5 (AUCCC) was established by nearest-neighbor analysis [8]. The identity of C6 (AUCCCC) and C7 (AUCCCCC) was inferred from steady-state transcription reactions carried out in the presence of limited NTPs, either the combination of [γ-32P]-ATP, UTP, and CTP, or the combination of ATP, UTP, and [α-32P]-CTP (results not shown).