5×amv rt reaction buffer  (Promega)

Journal: ACS Chemical Biology

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

doi: 10.1021/cb500270x

Figure Lengend Snippet: Incorporation of T opposite 7-EAA and 7-EAA triazole by AMV-RT reaction. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide (A, 7-EAA, or 7-EAA triazole). (B–D) Primer extension results for reactions containing different ddNTPs and different template strands, with N = A, 7-EAA, or 7-EAA-triazole.

Article Snippet: For each reaction, dNTPs, ddNTPs, 5× Promega AMV-RT buffer, and AMV-RT were added so that the concentrations in the reaction were as follows: 20 nM RNA, ∼80 nM 32 P-labeled 21 nt GluR B pre-mRNA primer, 10 mM of the 3 dNTPs, 10 mM of ddNTP for the fourth base, 1× Promega AMV-RT buffer, and 5 units of AMV-RT.

Techniques: Primer Extension Assay

Journal: ACS Chemical Biology

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

doi: 10.1021/cb500270x

Figure Lengend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Article Snippet: For each reaction, dNTPs, ddNTPs, 5× Promega AMV-RT buffer, and AMV-RT were added so that the concentrations in the reaction were as follows: 20 nM RNA, ∼80 nM 32 P-labeled 21 nt GluR B pre-mRNA primer, 10 mM of the 3 dNTPs, 10 mM of ddNTP for the fourth base, 1× Promega AMV-RT buffer, and 5 units of AMV-RT.

Techniques: Primer Extension Assay, Labeling, Inhibition, Standard Deviation