2.3 Buffers and
Reaction Mixes
- PCR mixture: 10μL N60 template DNA, 250μL2KOD FX
Neo buffer, 100μL dNTP mixture (2 mM each), 10μL10μM
Forward primer, 10μL10μM Reverse primer, 10μL 1.0 U/μ
L KOD FX Neo, and 110μL RNase-free water. - In vitro transcription mixture: 8μL template DNA, 2μL10
T7 reaction buffer, 2μL 2 mM ATP, 2μL 2 mM CTP, 2μL
2 mM GTP, and 2μL 2 mM UTP, 2μL T7 enzyme mix. - RT reaction mixture: 7μL template RNA, 0.5μL10μM
reverse primer, 2μL5RT reaction buffer, and 0.5μLRT
enzyme mix. - Ligation mixture: 5 ng insert DNA, 5 ng linearized plasmid
DNA, RNAse free water up to 1μL, and 1μL Ligation mix. - Annealing buffer: 10 mM Tris–HCl, pH 7.6, 100 mM KCl,
and 5 mM MgCl 2. - 2SSC buffer: 300 mM NaCl, 30 mM sodium citrate, pH 7.0,
50 v/v % formamide. - Prehybridization buffer: 300 mM NaCl, 30 mM sodium cit-
rate, pH 7.0, 10 w/v % dextran sulfate, 2 mM vanadyl-
ribonucleoside complex, 0.02 w/v % RNase-free BSA, 40μg
E. colitRNA, and 30 v/v % formamide.
2.4 Cell Culture Cells are maintained in Dulbecco’s modified Eagle medium, sup-
plemented with 100 units/mL penicillin, 100μg/mL streptomy-
cin sulfate, and 10 v/v % fetal bovine serum, at 37 Cina
humidified 5% CO 2 incubator.
- Trypsin–EDTA (0.25%), phenol red (e.g., Thermo Fisher
Scientific). - Dulbecco’s modified Eagle medium (e.g., Thermo Fisher
Scientific). - Penicillin–streptomycin, Liquid (10,000 units penicillin;
10,000μg streptomycin) (e.g., Thermo Fisher Scientific). - Fetal bovine serum (e.g., Biowest).
- Transfection Reagent (e.g., Promega FuGENE®HD).
- PDL-Coated Glass Bottom ViewPlate-96 (PerkinElmer).
- CO 2 incubator.
2.5 Equipment 1. HPLC system (e.g., Shimadzu Prominence).
- 5μm, 20.0100 mm preparative columns (e.g., GL Sciences
Inertsil ODS-3). - Fluorescence spectrometer (e.g., PerkinElmer LS 55).
- Spectrophotometer (e.g., Hitachi High-Technologies U-
3010). - Tabletop high-speed microcentrifuge.
308 Shin-ichi Sato et al.