Team:TU Darmstadt/Protocols

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Protocols

The following page(s) gives an overview of the protocols and possible modifications that where used and proven working during the iGEM PET.erminators project 2012. The corresponding materials can be found in the materials section.

Contents 1 In vivo 1.1 Escherichia coli 1.2 Comamonas testosteroni 2 In vitro 2.1 Nucleic acids 2.2 DNA 2.3 Protein 3 In silico 3.1 Data analysis 3.2 Databases 3.3 Modelling 3.4 Sequence analysis 3.5 Structure analysis 4 Material Science 5 Miscellaneous 5.1 Cell culture 5.2 Staining 5.3 ILDs / Improvised Lab Devices 5.4 Analytics

In vivo

Escherichia coli

• Bacterial cell culture
• Bacterial transformation aka TRAFO
• Cell counting/plating
• Chemically competent cells
• Colony PCR
• Electrocompetent cells
• Electroporation
• Glycerine Stocks
• Heat Shock Transformation
• Miniprep / Fermentas kit
• Miniprep / Promega kit
• Miniprep / Qiagen kit
• Protein Expression Est13
• Protein Expression FsC
• Western blot

Comamonas testosteroni

• Reviving a freez dried cell culture

In vitro

Nucleic acids

• Agarose gel electrophoresis
• PCR techniques
• SOE-PCR

DNA

• Agarose gel electrophoresis
• Ammonium acetate precipitation
• Cloning
• Dephosphorylation
• DNA Ligation
• DNA Ligation à la group transport
• DNA Quantification / NanoDrop
• Gel extraction
• Miniprep
• Mutagenic PCR
• PCR
• Primer Design
• Purification of DNA
• Restriction digest
• Resuspension of primers
• Salting Out
• Sequencing DNA

Protein

• Activity assay
• Affinity chromatography
• Immobilized metal ion affinity chromatography (IMAC)
• pNP-Assay Bacteria
• pNP-Assay Enzyme
• Proteinpurification
• Purification of Periplasmatic Proteins
• SDS-Page

In silico

Data analysis

• Information theory
• R Programming
• Mathlab / Simulink

Databases

• PDB
• CAS

Modelling

• Gaussian Network Modelling

Sequence analysis

• BLAST
• SAGE

Structure analysis

• PDB
• Protein structure prediction
• RNA secondary structure prediction

Material Science

• Synthesis of paranitrophenylesters with acyl chlorides in presence of triethylamine
• Synthesis of polyethylene terephthalate in presence of sulfuric acid
• Surface analysis of polyethylene terephthalate with atomic force microscopy

Miscellaneous

Cell culture

• Pouring LB plates

Staining

• Antibody staining

ILDs / Improvised Lab Devices

• x bp Primer Designer
• Enhancing a thermo cyclers cooling capabilites
• Gel pocket size reduction
• Growing iGEMlers

Analytics

• TEM-EDX Measurements
• GC-MS Measurements
• Preparation of Escherichia Coli for the energy dispersive X-ray spectroscopy (EDX)