Difference between revisions of "Team:TU-Eindhoven/Project/Protocols"
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<h1>General Protocols</h1> | <h1>General Protocols</h1> | ||
<h6>General Necessities | <h6>General Necessities | ||
| − | <br/><i>Preparation of antibiotics, growth media, stock solutions and more.</i><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Preparation of antibiotics, growth media, stock solutions and more.</i><br/> |
Glycerol Stock | Glycerol Stock | ||
| − | <br/><i>Preparing the bacteria for long term storage in the -80 °C freezer.</i><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Preparing the bacteria for long term storage in the -80 °C freezer.</i><br/> |
Streaking Glycerol Stock | Streaking Glycerol Stock | ||
| − | <br/><i>After storage in the -80 °C, the bacteria can be prepared for usage by streaking them on agar plates.</i><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>After storage in the -80 °C, the bacteria can be prepared for usage by streaking them on agar plates.</i><br/> |
Nanodrop | Nanodrop | ||
| − | <br/><i>Nanodrop is used to determine the concentration of a DNA sample.</i><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Nanodrop is used to determine the concentration of a DNA sample.</i><br/> |
Agarose Gel Electrophoresis | Agarose Gel Electrophoresis | ||
| − | <br/><i>Agarose Gel Electrophoresis is used to determine the length of DNA samples.</i><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Agarose Gel Electrophoresis is used to determine the length of DNA samples.</i><br/> |
SDS-Page | SDS-Page | ||
| − | <br/><i>SDS-Page is used to determine the size of a protein.</i></h6><br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>SDS-Page is used to determine the size of a protein.</i></h6><br/> |
<h1>Traditional Cloning & BioBricking</h1> | <h1>Traditional Cloning & BioBricking</h1> | ||
| − | <h6>PCR Amplification<br/> | + | <h6>PCR Amplification |
| − | PCR Purification<br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>PCR Amplification is used to amplify a DNA sequence.</i><br/> |
| − | Digestion<br/> | + | PCR Purification |
| − | Ligation<br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>PCR Purification is used to purify the product from a PCR reaction.</i><br/> |
| − | LIU PCR</h6><br/> | + | Digestion |
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>Digestion is used to cleave the plasmid and the insert in order to prepare them for ligation.</i><br/> | ||
| + | Ligation | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>Ligation is used to ligate a desired insert into a plasmid.</i><br/> | ||
| + | LIU PCR | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>LIU PCR is used to insert additional amino acids in a DNA sequence.</i><br/> | ||
| + | </h6><br/> | ||
<h1>Gibson Assembly</h1> | <h1>Gibson Assembly</h1> | ||
| − | <h6>Vector Linearization<br/> | + | <h6>Vector Linearization |
| − | < | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Vector Linearization is used to linearize a vector, after which it can be used for Gibson Assembly.</i><br/> |
| − | + | Gibson Assembly | |
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>The NEBuilder HiFi Assembly protocol is used for Gibson Assembly to ligate gBlocks into a linearized | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span>vector.</i><br/> | ||
| + | </h6><br/> | ||
<h1>Transformation, Protein Expression & Purification</h1> | <h1>Transformation, Protein Expression & Purification</h1> | ||
| − | <h6>Transformation into NovaBlue<br/> | + | <h6>Transformation into NovaBlue |
| − | Plating<br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>NovaBlue cells can be used for plasmid amplifcation. </i><br/> |
| − | Colony Picking and Colony PCR<br/> | + | Plating |
| − | Sequencing<br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Plating is required to amplify bacteria, by letting them grown on a growth medium.</i><br/> |
| − | Miniprep<br/> | + | Colony Picking and Colony PCR |
| − | Small culturing<br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Colony Picking and Colony PCR is used to evaluate if the inserted fragments have the correct length.</i><br/> |
| − | Protein expression<br/> | + | Sequencing |
| − | Protein Purification using a His-tag <br/> | + | <br/><span style="display:inline-block; width: 200px;"></span><i>Sequencing is used to determine the sequence of the amplified and transformed DNA.</i><br/> |
| − | Protein Purification using a Strep-tag</h6><br/> | + | Miniprep |
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>Miniprep is used to obtain the plasmid DNA.</i><br/> | ||
| + | Small culturing | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>Small culturing is used to amplify a specific bacteria colony.</i><br/> | ||
| + | Protein expression | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>This protocol is used to induce protein expression in bacteria from the small culture.</i><br/> | ||
| + | Protein Purification using a His-tag | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>This protocol is used to purify proteins using a His-tag.</i><br/> | ||
| + | Protein Purification using a Strep-tag | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i>This protocol is used to purify proteins using a Strep-tag. | ||
| + | </i><br/> | ||
| + | </h6><br/> | ||
<h1>Testing</h1> | <h1>Testing</h1> | ||
| − | <h6> | + | <h6>Flow chamber |
| − | Fluorescence | + | <br/><span style="display:inline-block; width: 200px;"></span><i>This protocol is used to prepare samples for microscopy.</i><br/> |
| + | Measuring Fluorescence | ||
| + | <br/><span style="display:inline-block; width: 200px;"></span><i></i><br/> | ||
| + | </h6><br/> | ||
Revision as of 14:25, 1 November 2017
General Protocols
General Necessities
Preparation of antibiotics, growth media, stock solutions and more.
Glycerol Stock
Preparing the bacteria for long term storage in the -80 °C freezer.
Streaking Glycerol Stock
After storage in the -80 °C, the bacteria can be prepared for usage by streaking them on agar plates.
Nanodrop
Nanodrop is used to determine the concentration of a DNA sample.
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis is used to determine the length of DNA samples.
SDS-Page
SDS-Page is used to determine the size of a protein.
Traditional Cloning & BioBricking
PCR Amplification
PCR Amplification is used to amplify a DNA sequence.
PCR Purification
PCR Purification is used to purify the product from a PCR reaction.
Digestion
Digestion is used to cleave the plasmid and the insert in order to prepare them for ligation.
Ligation
Ligation is used to ligate a desired insert into a plasmid.
LIU PCR
LIU PCR is used to insert additional amino acids in a DNA sequence.
Gibson Assembly
Vector Linearization
Vector Linearization is used to linearize a vector, after which it can be used for Gibson Assembly.
Gibson Assembly
The NEBuilder HiFi Assembly protocol is used for Gibson Assembly to ligate gBlocks into a linearized
vector.
Transformation, Protein Expression & Purification
Transformation into NovaBlue
NovaBlue cells can be used for plasmid amplifcation.
Plating
Plating is required to amplify bacteria, by letting them grown on a growth medium.
Colony Picking and Colony PCR
Colony Picking and Colony PCR is used to evaluate if the inserted fragments have the correct length.
Sequencing
Sequencing is used to determine the sequence of the amplified and transformed DNA.
Miniprep
Miniprep is used to obtain the plasmid DNA.
Small culturing
Small culturing is used to amplify a specific bacteria colony.
Protein expression
This protocol is used to induce protein expression in bacteria from the small culture.
Protein Purification using a His-tag
This protocol is used to purify proteins using a His-tag.
Protein Purification using a Strep-tag
This protocol is used to purify proteins using a Strep-tag.
Testing
Flow chamber
This protocol is used to prepare samples for microscopy.
Measuring Fluorescence
