Selected article for: "reaction buffer and Tris HCl"
Author: Lifeng Zhou; Arun Richard Chandrasekaran; Jibin Abraham Punnoose; Gaston Bonenfant; Stephon Charles; Oksana Levchenko; Pheonah Badu; Cassandra Cavaliere; Cara T. Pager; Ken Halvorsen
Title: Programmable low-cost DNA-based platform for viral RNA detection
  • Document date: 2020_1_16
    • ID: 8kced06y_34
    Snippet: First, we employed the classic NASBA protocol 41 to prove the concept (Fig. S15) . The 25 µl one-pot reaction contained 3 µl RNA sample at various concentrations, 0.4 µM forward and reverse primers, 8 U AMV Reverse Transcriptase, 50 U T7 RNA Polymerase, 0.1 U RNase H, 40 U RNase Inhibitor (NEB, Murine), 2 mM NTP mix, 1 mM rNTP mix, 12 mM MgCl 2 , 40 mM Tris-HCl, 42 mM KCl, 5 mM Dithiotreitol (DTT), 15%(v/v) dimethyl sulfoxide. The primers were.....
    Document: First, we employed the classic NASBA protocol 41 to prove the concept (Fig. S15) . The 25 µl one-pot reaction contained 3 µl RNA sample at various concentrations, 0.4 µM forward and reverse primers, 8 U AMV Reverse Transcriptase, 50 U T7 RNA Polymerase, 0.1 U RNase H, 40 U RNase Inhibitor (NEB, Murine), 2 mM NTP mix, 1 mM rNTP mix, 12 mM MgCl 2 , 40 mM Tris-HCl, 42 mM KCl, 5 mM Dithiotreitol (DTT), 15%(v/v) dimethyl sulfoxide. The primers were chosen from reference 21 . The sample was incubated at 41 °C for 2 hours in the thermal cycler and followed by heating at 94 °C for 10 minutes to deactivate all enzymes. Three microliters of the sample were used in the following DNA nanoswitch detection assay in PCR tubes with 10 µl final volumes. After mixing with the DNA nanoswitch and reaction buffer, the mix was incubated at room temperature for two hours. GelRed (Biotium Inc.) at 1×concentration was added to the detection samples before loading to the 0.8% agarose gel. The gel was run at room temperature for 45 minutes at 75 volts.

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