"
Team:EPF-Lausanne/Protocols
From 2011.igem.org
(Difference between revisions)
(→Cloning, assembly, and mutations) |
(→Cloning, assembly, and mutations) |
||
| (13 intermediate revisions not shown) | |||
| Line 7: | Line 7: | ||
* [[Team:EPF-Lausanne/Protocols/Competent cells|Competent Cells]]: prepare cells for plasmid uptake. | * [[Team:EPF-Lausanne/Protocols/Competent cells|Competent Cells]]: prepare cells for plasmid uptake. | ||
* [[Team:EPF-Lausanne/Protocols/Competent cells. Protocol II|Competent Cells. Protocol II (Inoue method)]]. | * [[Team:EPF-Lausanne/Protocols/Competent cells. Protocol II|Competent Cells. Protocol II (Inoue method)]]. | ||
| - | * [[Team:EPF-Lausanne/Protocols/Glycerol stock|Glycerol stock]]: stock transformed cells at -80°C | + | * [[Team:EPF-Lausanne/Protocols/Glycerol stock|Glycerol stock]]: stock transformed cells at -80°C. |
| + | * [[Team:EPF-Lausanne/Protocols/Agar Plates|Agar plate preparation]]: preparing agar plates for cell cultures. | ||
| + | * [[Team:EPF-Lausanne/Protocols/Autoclave|Autoclave]]: to sterilize solutions and glassware. | ||
=== Cloning, assembly, and mutations === | === Cloning, assembly, and mutations === | ||
| Line 17: | Line 19: | ||
* [[Team:EPF-Lausanne/Protocols/TetR|Linear template- TetR]]. | * [[Team:EPF-Lausanne/Protocols/TetR|Linear template- TetR]]. | ||
* [[Team:EPF-Lausanne/Protocols/TetR Extension PCR|tetR Overlap Extension PCR]]: induce specific mutations on tetR linear template. | * [[Team:EPF-Lausanne/Protocols/TetR Extension PCR|tetR Overlap Extension PCR]]: induce specific mutations on tetR linear template. | ||
| + | * [[Team:EPF-Lausanne/Protocols/Site-specific mutagenesis|tetR Site-specific mutagenesis]]: induce site-specific mutations on a plasmid containing tetR. | ||
* [[Team:EPF-Lausanne/Protocols/Gel_Electrophoresis|Agarose gel electrophoresis]]: DNA samples analysis (can be followed by band purification). | * [[Team:EPF-Lausanne/Protocols/Gel_Electrophoresis|Agarose gel electrophoresis]]: DNA samples analysis (can be followed by band purification). | ||
| + | * [[Team:EPF-Lausanne/Protocols/Colony PCR|Colony PCR]]: Analyze the plated transformed cells, to see if Gibson assembly worked | ||
| + | * [[Team:EPF-Lausanne/Protocols/T7-ext|T7 extension PCR]]: Put T7 promoter upstream of a gene | ||
| + | * [[Team:EPF-Lausanne/Protocols/bluntends|Blunt-End Cloning]]: Making Blunt Ends | ||
=== DNA recovery === | === DNA recovery === | ||
| Line 23: | Line 29: | ||
* [[Team:EPF-Lausanne/Protocols/Miniprep|Plasmid preparation - Miniprep]]: recover plasmids from a bacterial culture. | * [[Team:EPF-Lausanne/Protocols/Miniprep|Plasmid preparation - Miniprep]]: recover plasmids from a bacterial culture. | ||
* [[Team:EPF-Lausanne/Protocols/Gel purification|Gel purification]]: recover DNA separated by electrophoresis | * [[Team:EPF-Lausanne/Protocols/Gel purification|Gel purification]]: recover DNA separated by electrophoresis | ||
| + | |||
| + | == Biochemistry == | ||
| + | * [[Team:EPF-Lausanne/Protocols/IPTG test|IPTG test]]: test expression of a gene downstream from Plac | ||
== Microfluidics == | == Microfluidics == | ||
* [[Team:EPF-Lausanne/Protocols/PDMS_two_layer_device_fabrication|PDMS two layer device fabrication]] | * [[Team:EPF-Lausanne/Protocols/PDMS_two_layer_device_fabrication|PDMS two layer device fabrication]] | ||
| + | * [[Team:EPF-Lausanne/Protocols/Master microfabrication for PDMS replica molding |Master microfabrication for PDMS replica molding]] | ||
* [[Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA|MITOMI: Protein – DNA interactions]] | * [[Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA|MITOMI: Protein – DNA interactions]] | ||
| - | |||
* [[Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis|Klenow dsDNA synthesis]] | * [[Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis|Klenow dsDNA synthesis]] | ||
| + | |||
| + | == Worm culture == | ||
| + | * [[Team:EPF-Lausanne/Protocols/MG_Agar_Plates|Agar plate preparation]]: preparing MG Agar plates for worm cultures. | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Latest revision as of 15:35, 16 October 2011
Protocols
Contents |
Molecular Biology
Products and stock preparation
- Primer preparation: initial dilution of ordered primers.
- Competent Cells: prepare cells for plasmid uptake.
- Competent Cells. Protocol II (Inoue method).
- Glycerol stock: stock transformed cells at -80°C.
- Agar plate preparation: preparing agar plates for cell cultures.
- Autoclave: to sterilize solutions and glassware.
Cloning, assembly, and mutations
- Transformation: introduce foreign plasmid into competent cells.
- Plating: plate transformed cells
- Liquid cultures: when cells have grown on plates, put them in liquid cultures
- Gibson assembly.
- Linear template- TetR.
- tetR Overlap Extension PCR: induce specific mutations on tetR linear template.
- tetR Site-specific mutagenesis: induce site-specific mutations on a plasmid containing tetR.
- Agarose gel electrophoresis: DNA samples analysis (can be followed by band purification).
- Colony PCR: Analyze the plated transformed cells, to see if Gibson assembly worked
- T7 extension PCR: Put T7 promoter upstream of a gene
- Blunt-End Cloning: Making Blunt Ends
DNA recovery
- Plasmid preparation - Miniprep: recover plasmids from a bacterial culture.
- Gel purification: recover DNA separated by electrophoresis
Biochemistry
- IPTG test: test expression of a gene downstream from Plac
Microfluidics
- PDMS two layer device fabrication
- Master microfabrication for PDMS replica molding
- MITOMI: Protein – DNA interactions
- Klenow dsDNA synthesis
Worm culture
- Agar plate preparation: preparing MG Agar plates for worm cultures.
