| LR Reaction |
| 1. | Mix the following. |
|
| Component |
|
| LR reaction mixture | 5.00 µl |
| Entry clone (2 ng/µl) | 1.00 µl |
| Destination vector (20 ng/µl) | 0.50 µl |
| LR Clonase enzyme mix (Invitrogen) | 0.25 µl |
| 5×LR Clonase buffer (Invitrogen) | 1.00 µl |
| TE buffer | 2.25 µl |
|
| 2. | Incubate reactions at 25°C for 18 h. |
|
|
| PCR |
| 1. | Mix the following. |
|
| Component |
|
| PCR reaction mixture | 50.00 µl |
| Distilled water | 30.75 µl |
| dNTPs (2 mM) | 5.00 µl |
| 10×PCR buffer | 5.00 µl |
| DMSO | 5.00 µl |
| pEu3H-F-primer (10 µM) | 1.00 µl |
| pEu3H-R-primer (10 µM) | 1.00 µl |
| KOD Dash (2.5 units/µl) (TOYOBO) | 0.25 µl |
| LR reaction mixture | 2.00 µl |
|
| 2. | Perform PCR reactions according to the following cycling parameters: |
| 95°C×1 min; [94°C×20 sec, 55°C×5 sec, 74°C×4 min]×30 cycles. |
|
|
| mRNA Synthesis |
| 1. | Mix the following. |
|
| Component |
|
| mRNA synthesis reaction mixture | 50.0 µl |
| 5×TB buffer | 10.0 µl |
| NTPs mix (25 mM) | 5.0 µl |
| RNase inhibitor (40 units/µl) (TOYOBO) | 1.0 µl |
| SP6 RNA polymerase (50 units/µl) (TaKaRa) | 3.0 µl |
| PCR reaction mixture | 5.0 µl |
| Sterile MilliQ | 26.0 µl |
|
| 2. | Incubate reactions at 37°C for 18 h. |
|
|
| Protein Synthesis (RI-Bilayer Method) |
| 1. | Mix the following for each layer. |
| < Lower layer >
| Component |
|
| Total | 50.0 µl | | †1 Supplied by a manufacturer |
| Buffer #2 †1 | 2.0 µl |
| 4×Buffer Mix †1 | 0.5 µl |
| Creatine kinase (10 mg/ml) †1 | 1.7 µl |
| RNase inhibitor (40 units/µl) | 1.0 µl |
| Wheat germ extract (TOYOBO) | 10.0 µl |
| 14C-Leu (GE Healthcare) | 606 µl |
| mRNA mixture (0.5-0.75 µg/µl) | 28.2 µl |
|
| < Upper layer >
| Component |
|
| Total | 250 µl | | †1 Supplied by a manufacturer |
| Buffer Mix †1 | 250 µl |
|
| 2. | Incubate reactions at 26°C for 18 h. |
|
|
| Electrophoresis |
| 1. | Dispense the reaction mixture (130 µl) into 1.5 ml microtube. |
| 2. | Centrifuge at 19,000×g for 20 min at 4°C. |
| 3. | Mix the reaction mixture (3 µl) or the supernatant (3 µl) with 4×SDS-sample buffer (2 µl). |
| 4. | Heat at 70°C for 10 min. |
| 5. | Perform SDS-PAGE under the following. |
|
| Conditions |
| • Gel : NuPAGE 4-12% Bis-Tris Gel (1.0 mm, 17 well) (Invitrogen) |
| • Migration buffer : NuPAGE MES SDS Running Buffer (Invitrogen) |
| • Add NuPAGE Antioxidant (0.5 ml) (Invitrogen) into buffer of the negative electrode. |
| • Marker : MagicMark Western Protein Standard (Invitrogen) |
| • Applied sample volume : 8 µl |
| • Electrophoresis : 200 V (constant) and 2 A for 47 min |
|
|
|
| Detection |
| 1. | Fix of the gel by 20% TCA for 1 h and remove free RI-labeled amino acids. |
| 2. | Dry the gel. |
| 3. | Analyse with BAS2000 (Fuji). |
|
|