Protocols

Here is a list of all the procedures we used this summer. Each contains a description and list of materials required.

General Protocols

• Agarose Gel Electrophresis
• Bacterial Genomic DNA Purification
• Bacterial Transformation
• Construction Techniques
• GC-MS Analysis
• Gel Extraction
• Gibson Assembly
• LB Agar Plates
• M9 Minimal Media Preparation
• Organic Extraction
• Overnight Cultures
• PCR Purification
• PicoGreen Assay
• Plasmid Purification (from E. coli)
• Preparing Chemically Competent Cells (E. coli)
• Preparing Glycerol Stocks (E. coli)
• Rehydration of Registry DNA
• Reviving Freeze-dried Bacterial Cultures from DSMZ
• Site-Directed Mutagenesis
• Splice Overlap Extension PCR (SOE PCR)

Electrochemistry Protocols

• Cyclic Voltammetry
• Potentiostatic Standard Curve Generation
• Reporter Expression Detection

Desulfurization Protocols

• Catalase assay
• Desulfurization Assay
• HpaC assay

Decarboxylation Protocols

• PetroBrick Validation Assay
• oleT Validation Assay

Denitrogenation Protocols

• AmdA Characterization Assay
• Carbazole Degradation Assay

Decatecholization Protocols

• Decatecholization Assay

Transposon Mutant Library for Toxin Detection

• Transposon-Mediated Mutant Library Generation

Kill Switch Protocols

• Characterization of mgtA regulation with GFP
• Characterization of mgtA regulation with S7 killgene
• Characterization of P_rha with GFP
• Kill Assay for rhamnose system
• Glycine Auxotrophic Assays (Glycine Media Test, and Petrobrick Test)
• Nuclease assay

Bioreactor Assays Protocols

• Bacterial Growth Experiments

Belt Selection Tests:

• Bacteria Test
• Hydrocarbon Separation Test
• Hydrocarbon Test
• NA and Hexadecane Belt Skim Test
• Tailings Test

Modeling Protocols

• Modelling validation experiments