Team:TU Darmstadt/Protocols

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 [http://en.wikipedia.org/wiki/E._coli Escherichia coli] 1.2 Comamonas testosteroni 2 In vitro 2.1 Nucleic acids 2.2 [http://en.wikipedia.org/wiki/DNA DNA] 2.3 Protein 3 Material Science 4 Miscellaneous 4.1 Staining 4.2 Analytics 4.3 ILDs / Improvised Lab Devices

In vivo

[http://en.wikipedia.org/wiki/E._coli 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

• [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf Reviving a freez dried cell culture]

In vitro

Nucleic acids

• Agarose gel electrophoresis
• PCR techniques
• SOE-PCR

[http://en.wikipedia.org/wiki/DNA DNA]

• Agarose gel electrophoresis
• Ammonium acetate precipitation
• Cloning
• Dephosphorylation
• DNA Ligation
• DNA Ligation à la group transport
• DNA Quantification / NanoDrop
• Miniprep
• Mutagenic PCR
• PCR
• Purification of DNA
• Restriction digest
• Salting Out
• [http://www.eurofinsdna.com/de/home.html?wmc=GA_DE-Corporate&gclid=CODAs8CZ1LICFQVf3god9AIAag 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

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

Staining

• Antibody staining

Analytics

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

ILDs / Improvised Lab Devices

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