Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
Figure Lengend Snippet: Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.
Article Snippet: Ligation reactions contained 10 pmol of synthetic ssRNA oligonucleotides (21-mer) either with 2′-hydroxyl or 2′- O -methyl 3′-ends, 20 pmol of pre-adenylated DNA adapter (Universal miRNA Cloning Linker; New England Biolabs Inc.) in 1× reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT at pH 7.5, 25°C) in 20-μL final reaction volumes.
Techniques: Ligation, Staining, Incubation, Concentration Assay