Typhoon fla 7000 fluorescence image analyzer

RNA aminoacylated with Phe^AZ-CME and clicked with DBCO-sulfo-biotin was precipitated by ethanol and dissolved in SAv loading buffer (0.5 mM sodium acetate (pH = 5.2), 4 M urea, 1 mM Na₂EDTA, 1 mM Tris, 1.67 mg/ml streptavidin (Z7041, Promega)). The resulting solution was loaded on denaturing PAGE (8 M urea, 1× TBE). After electrophoresis, gels were stained with ethidium bromide and imaged with Typhoon FLA 7000 fluorescence image analyzer (GE Healthcare). Aminoacylation efficiency was determined from the band intensity of SAv-binding RNAs (S) and free RNA (F) and calculated as S/(S + F).

0.75 μM oligo 23 was labeled with ³²P by T4 polynucleotide Kinase (2021A, TaKaRa) in the presence of adenosine 5′-triphosphate, [γ-³²P] (6000 Ci/mmol, 10 mCi/ml, NEG502Z, Perkin Elmer), following the manufacturer's protocol. 5′-³²P labeled primer was purified by denaturing PAGE containing 8 M urea and 1× TBE. Purified primer was stocked in 1 mM HEPES-KOH (pH = 8.0).
inT1-at-s was first aminoacylated in the presence or absence of Phe^AZ-CME, precipitated by ethanol, and resuspended in 0.5× TE buffer (5 mM Tris, 0.5 mM EDTA, pH = 7.4). Both inT1-at-s samples were reverse transcribed by Superscript IV (18090050, Invitrogen) following the protocol described in (53 (link)), while the concentration of each dNTP was adjusted to 10 μM to induce reverse transcription stop.
For the ladders to identify the reverse transcription stop site, non-aminoacylated inT1-at-s was reverse transcribed by Superscript III (18080044, Invitrogen) following the protocol described in (53 (link)). In each entry, 1 mM of ddATP, ddTTP, ddCTP or ddGTP was added to the reaction to stop reverse transcription randomly.
Reactions were quenched and loaded onto denaturing PAGE (7 M urea, 1 × TBE) and run at 50 W for 4 h. After electrophoresis, the gel was exposed to an IP cassette (Fujifilm) and imaged by Typhoon FLA 7000 fluorescence image analyzer (GE Healthcare).

The translation mixture was performed in the presence of 50 μM l-[¹⁴C(U)]-Aspartic acid (>200 mCi/mmol, 0.1 mCi/ml, NEC268E, Perkin Elmer). After translation, the solution was mixed with 2x tricine SDS-PAGE loading buffer (0.9 M Tris–HCl (pH = 8.45), 8% SDS, 30% glycerol, and 0.001% xylene cyanol) and subjected to 15% tricine SDS-PAGE gels at 150 V for 40 min. The resulting gel was exposed to an IP cassette (Fujifilm) and imaged by Typhoon FLA 7000 fluorescence image analyzer (GE Healthcare).