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Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE

Home > Products > First Strand Synthesis Protocol with Reverse Transcriptase First Strand Synthesis Protocol with Reverse Transcriptase Overview Materials needed: 10X RT buffer: 500 mM Tris-HCl (pH 8.3 @ 25°C) 750 mM KCl 30 mM MgCl2 100 mM DTT dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0) Oligo-dT primer (40 µM) Random nonamers (40 µM) RNase Inhibitor (10 U/µL) M-MuLV Reverse Transcriptase (200 units/µL) Introduction Protocol In a sterile microfuge tube add: RNA solution  0.5-2 µg ( total RNA or 50-100 ng polyA-selected RNA) Primer (dT or N9)         2 µL dNTP mix                     4 µL nuclease-free H2O to final volume of 16 µL Heat for 3-5 minutes at 65-80°C. Spin briefly and place promptly on ice. Add: 10X RT buffer 2 µL RNAse inhibitor 1 µL M-MuLV Reverse Transcriptase 1 µL ______ final volume 20 µL Incubate at 42°C for one hour. Inactivate enzyme at 90°C for 10 minutes. Store products at -20°C or proceed to next step(s).