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Figure 3 | Biological Procedures Online

Figure 3

From: Increasing the library size in cDNA display by optimizing purification procedures

Figure 3

Optimization of the amount of SA-beads to bind the mRNA-protein fusion molecules. (A) mRNA-protein fusion was prepared from DNA template coding B domain of protein A (BDA) using SBP-linker as described Figure 1. Translation mixture was treated with EDTA same as Figure 2. Then the mixture was incubated with 0.0075–0.2 mg of SA-beads per 0.5 pmol of mRNA estimated from the amount in the ligation reaction [the ratio of the biotin-binding capacity of the SA-beads to the total amount of SBP-linker (containing a single biotin) is 7.5–200]. Inputted mRNA-linker conjugates (lane I), translation mixture (lane T), and remaining mRNA-protein fusion molecules in the translation mixture after incubation with different amounts of SA-beads were analyzed by 4% stacking–6% separating SDS-PAGE containing 8 M urea. (B) Binding efficiencies of each ratio were calculated by: [band intensity of the mRNA-protein fusion molecules in lane T] - [band intensity of the remaining mRNA-protein fusion molecules in each lane indicated by SA-beads]. Experiments were repeated 3 times. Error bars = standard deviation. (C) mRNA/cDNA-protein fusion molecules were prepared with each ratio of SA-beads. Reverse transcription (RT) was performed at 40°C for at least 10 min in 20 μL of the RT reaction mixture [50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 50 mM dithiothreitol (DTT), 0.5 mM dNTP mix and 200 U of SuperScriptIII reverse transcriptase (Invitrogen)] . 20 U of RNase T1 (Ambion) and RNase H (Takara Bio Inc., Kyoto, Japan) were added to the RT reaction mixture and incubated at 37°C for 10 min. Released mRNA/cDNA-protein fusion molecules were confirmed by SDS-PAGE as the above. (D) The amount of cDNA-protein fusion molecules calculated by comparing the band intensity between the cDNA-protein fusion molecule of each lane and 0.5 pmol of mRNA-linker conjugate.

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