1) Overview
This kit is a T7 promoter–driven prokaryotic cell-free expression system. It efficiently expresses prokaryotic proteins (including bacterial and viral proteins; supports proteins with disulfide bonds) and eukaryotic proteins that do not require post-translational modifications. A set of positive-control plasmids (e.g., GFP, human IgG, and a membrane protein) is recommended to validate performance—especially if you are new to in-vitro transcription/translation.
2) General Handling & Safety
- Work RNase-free: wear gloves; use nuclease-free tubes, tips, and water.
- Keep all reagents on ice during reaction setup; avoid repeated freeze–thaw.
- Mix thoroughly by gentle pipetting before use; avoid bubbles/foaming.
- Typical reaction volume is 50 μL (can be scaled up or down as needed).
3) Template Requirements & Yield Determinants
Use DNA templates designed for T7 transcription with an appropriate ribosome binding site (RBS).
Protein yield depends on:
- Protein size (molecular weight)
- DNA template quality and purity
- Coding sequence features
- Gene position relative to the T7 promoter and RBS
E. coli codon optimization is strongly recommended. Without it, expression may be low or even undetectable for some genes.
4) Reaction Setup (50 μL standard)
Thaw on ice the required aliquots of Buffer A and Buffer B. Quick-spin to collect contents.
Assemble on ice in a new tube in this order (per 50 μL reaction):
Reagent Volume
Buffer A 20.5 μL
Buffer B 16.7 μL
DNA template (final 20 ng/μL) X μL
Nuclease-free H₂O (12.8 − X) μL
Total 50.0 μL
- Mix gently by pipetting (do not vortex).
- Strongly recommended: add RNase Inhibitor per 50 μL reaction, especially plasmids prepared by mini-prep plasmid DNA (e.g. Qiagen).
- Tip: A practical starting DNA amount is 1–3 μL of a 20 ng/μL template (final 20–60 ng per 50 μL).
5) Incubation (Protein Synthesis)
Choose one of the following:
- Shaker/incubator: 25 °C, ~180 rpm
- Thermomixer: 25 °C, ~600 rpm
Time:
- Many proteins reach near-maximal yield in 6–8 h.
- 2–16 h is the workable range; some targets benefit from overnight (16 h).
- For first-time targets, start with 16 h to ensure complete expression, then optimize.
6) Vessel Selection & Scaling
Use flat-bottom cell-culture plates or flasks (recommended). Deep-well plates are not recommended.
Reaction Volume Recommended Vessel
50 μL 96-well plate
100 μL 48-well plate
200–300 μL 24-well plate
500–1000 μL 6-well plate
10–100 mL 250 mL baffled or conical flask
7) Post-Reaction Processing
- Centrifuge the reaction at 4 °C, 10,000 × g, 2 min.
- Collect supernatant for analysis (retain pellet if you need to check insoluble fraction).
Analysis options: SDS-PAGE, Western blot, or target-specific functional assays. GFP control can be monitored by fluorescence.
8) Positive Controls (Recommended Use)
- GFP plasmid: quick visual/fluorescent readout of expression performance.
- Human IgG plasmid: tests expression of multi-domain, higher-MW proteins.
- Membrane protein plasmid: validates workflow for challenging targets.
9) Storage & Reagent Management
- Store kit components at –80 °C.
- Avoid multiple freeze–thaw cycles.
- For batching: you may premix one tube of Buffer A with one tube of Buffer B, aliquot on ice, snap-freeze in liquid nitrogen, and store at –80 °C for later use.
10) Troubleshooting & Optimization (Quick Guide)
- Low or no expression: verify T7 promoter/RBS placement; improve DNA purity; codon-optimize; extend incubation to 16 h.
- RNase issues (mRNA degradation): switch to high-quality plasmid/prep; add RNase inhibitor; re-check RNase-free technique.
- Target too large/complex: test N-/C-terminal tags; evaluate domain constructs; consider lowering or mildly increasing temperature (e.g., 22–30 °C) in small tests.
- Foaming/bubbles: reduce pipetting force; do not vortex; use wide-bore tips for viscous mixes.
- Between-well variability: confirm plate is flat-bottom; ensure uniform shaking speed and temperature; pre-equilibrate instruments.
11) Critical Notes
- Setups must remain on ice to prevent premature activity or component degradation.
- Minimize bubbles—they can impair mixing/oxygen transfer and reduce yield.
- DNA quality matters—mini-prep plasmids may carry RNase; RNase inhibitor is strongly recommended.
- Actual yields vary by gene size/sequence and promoter–RBS spacing; expect to empirically optimize these parameters for best performance.