RNasinRibonuclease Inhibitor (Promega)
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Reverse transcription (RT) reactions (40 [micro]L) were performed at 42 [degrees] C for 45 min in the presence of 2 [micro]g total RNA, 1x first strand buffer as supplied by the manufacturer, dNTPs to 1 mM each, 2 [micro]M primer, 1 mM dithiothreitol, 40 units of RNasin (Promega Corp.
5 [micro]M oligo(dT)18, 200 [micro]M dNTPs, 10 U RNAsin (Promega, Madison, WI, USA), 1 x M-MLV RT buffer, and 300 U M-MLV reverse transcriptase (Promega, USA) in a volume of 50 pL at 40[degrees]C for 1 h.
2]O (2ng/[micro]L) for reverse transcription with the addition of 5 [micro]L of a reaction mixture containing 5X first strand buffer, 10 mmol/L deoxynucleoside-5'-triphosphate, RNasin, Moloney murine leukemia virus reverse transcriptase, and miR-specific RT primers (Exiqon).
5 mM concentrations of dATP, dCTP, dGTP, and dUTP; 20 units of RNasin (Invitrogen), 1 [micro]L T7 RNA polymerase (Invitrogen), 0.
1] XDNA oligo (5'-GAG AGA CTC GAG AGA GAC CGT CTA CGA ATT CTG CAT C (T)24-3'), and RNasin (20 units).
6 mmol/L deoxynucleoside triphosphates, 1 [micro]mol/L of each primer, 20 U Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA), 4 U Recombinant RNAsin ribonuclease inhibitor (Promega), and 1 U DyNAzyme (Finnzymes, Espoo, Finland).
3 units/[mu]L RNAsin ribonuclease inhibitor (Promega Corp, Madison, WI, USA), 7-5 [micro]M random primers [oligo(dN)6], 1.
1] nucleotide triphosphates (MBI Fermentas), 12 [micro]l of RNAsin (Promega), 15 [micro]l of T7 RNA polymerase (MBI Fermentas), and distilled water (d[H.