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 [http://en.wikipedia.org/wiki/RNA RNA] 2.4 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 Microscopy 5.3 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
• Engineering BioBrick vectors from BioBrick parts/Colony PCR
• Heat Shock Transformation
• In-fusion BioBrick assembly
• Miniprep / Fermentas kit
• Miniprep / Promega kit
• Miniprep / Qiagen kit
• Transformation of chemically competent cells
• 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 sulfate precipitation
• Annealing and primer extension
• Annealing primers
• Assembly PCR
• Cloning
• DNA Ligation
• DNA Quantification / NanoDrop
• DNA Synthesis from Oligos
• Engineering BioBrick vectors from BioBrick parts/Colony PCR
• Miniprep
• PCR
• Polyacrylamide gel electrophoresis
• Primer Design
• Purification of DNA
• Restriction digest
• Resuspension of primers
• Salting Out
• Sequencing DNA

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

Protein

• Activity assay
• Affinity chromatography
• Immobilized metal ion affinity chromatography (IMAC)
• 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

Miscellaneous

Cell culture

• Pouring LB plates

Microscopy

• Antibody staining
• Determining an objects size
• Preparing slides
• Staining

ILDs / Improvised Lab Devices

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

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