Fig. 1. Minimizing 3′ heterogeneity in T7 transcripts. (A) Plasmid templates (see Materials and methods) were digested with the indicated restriction enzymes and transcribed in mixtures containing 2, 3 or 8 mM MgCl₂. Sample loading was adjusted to approximately equalize the amount of N-length RNA in each lane. The fraction of N-length RNA is indicated below each panel. (B) Quantification of N + 1 (and longer) RNAs as a percentage of total RNA in transcription reactions of RS19 (circles) or RZ6 (triangles) templates in reactions containing 2–25 mM MgCl₂. (C) Time course of synthesis of RS19 RNA in various concentrations of MgCl₂. The yields in PhosphorImager (PI) counts of total RNA (open symbols) and N-length RNA (filled symbols) from reactions containing 2, 3 and 8 mM MgCl₂ are indicated by circles, triangles and squares, respectively. (D) DEPC structure probing of 3′ end-labeled G11 D RNA synthesized with 2 or 8 mM MgCl₂. RNAs were partially modified with DEPC at 37°C in 200 mM HEPES pH 8.0, 50 mM KCl, ±20 mM MgCl₂ or in denaturing (Den) conditions (200 mM HEPES pH 8.0, 1 mM EDTA, 90°C). Base numbers are as in Beattie et al. (1995).