Experimental Techniques

Synthetic biology focuses on data quantification and precise phenotypic characterization. Protein electrophoresis, nucleic acid detection, enzyme activity detection, and metabolite quantification are the core detection methods. The key is to avoid experimental errors and ensure the authenticity and repeatability of the data.

1. Techniques for nucleic acid detection (PCR/ agarose gel electrophoresis)

• Standardization of gel preparation: The concentration of nucleic acid electrophoresis gel is adjusted according to the requirements: 1% gel is used for the detection of 1000–5000 bp long fragments, 1.5% gel for 200–1000 bp medium fragments, and 2% gel for fragments shorter than 200 bp, ensuring band resolution.

• Control of electrophoresis conditions: Constant voltage electrophoresis, 80 V during the concentration stage, 120 V during the separation stage. Avoiding high voltage leads to band dispersion, and low voltage leads to band tailing; the sample volume is uniformly 5–10 μL, excess leads to band accumulation, and insufficient leads to blurred bands that cannot be observed.

• Avoiding imaging pitfalls: Imaging immediately after gel staining to avoid fading of bands due to long exposure; uniform background calibration during imaging to ensure accurate quantitative gray-scale, which can be used for subsequent analysis of amplification efficiency and fragment purity of elements.

2. Techniques for protein SDS-PAGE electrophoresis detection

• Sample pre-treatment standardization: The ratio of protein sample to loading buffer is 4:1, boiled for 10 minutes in boiling water bath to fully denature the protein, avoiding protein aggregation that leads to band tailing and increased background bands; quickly centrifuge after boiling, take the supernatant for loading, eliminating interference from impurities.

• Control of electrophoresis layering: Stable voltage of 80 V for the浓缩 gel， sample pressure to the separation gel interface and then adjusted to 120 V， ensuring flat bands without inclination throughout the process； adjust the electrophoresis time according to the molecular weight of the target protein to prevent small molecule proteins from running out of the gel.

• Optimization of staining and decolorization： 1-hour Coomassie Brilliant Blue staining， decolorization until the background is transparent and the bands are clear， excessive decolorization can lead to the disappearance of faintly expressed bands； throughout the process， avoid gel contamination to ensure that the experimental results can be photographed for retention and quantitative analysis.