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Team:Heidelberg LSL/Notebook methods
From 2012hs.igem.org
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<h2>Methods</h2> | <h2>Methods</h2> | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
| - | <p>...<br/> | + | <p>Transformation is a process in which linear or circular DNA, in other words your designed plasmid, is brought into competent bacteria without the help of bacteriophages. You do this mostly with one of these two intentions: firstly, you want to amplify of your chosen Biobrick-DNA and let transformed bacteria grow it for you is the cheapest and fastest way. Afterwards you have to extract your chosen Biobrick out of your bacteria. Secondly, when you completed your construct in form of a plasmid, you might need to bring it into your final bacteria cells to test if it works.<br/> |
| + | For amplifying DNA from the registry, we transformed chemocompetent Top10 cells by applying the following protocol: <br/> | ||
| + | DNA from Registry Plate: 10 ul water is added to the corresponding well containing the desired BioBrick DNA and DNA is solved by incubation for 15 min at room temperature. 5 ul of DNA solution are added to 50 ul of chemocompetent Top10 cells and incubated on ice for 20 min. Afterwards, a heat shock is performed by incubation the bacteria on 42°C/50 s. After another 2 min on ice, the bacteria are plated onto LB-Amp agar plates.<br/> | ||
<h4>Inoculation of LB agar plates</h4> | <h4>Inoculation of LB agar plates</h4> | ||
<p>...<br/> | <p>...<br/> | ||
Revision as of 19:24, 11 June 2012
Methods
Transformation
Transformation is a process in which linear or circular DNA, in other words your designed plasmid, is brought into competent bacteria without the help of bacteriophages. You do this mostly with one of these two intentions: firstly, you want to amplify of your chosen Biobrick-DNA and let transformed bacteria grow it for you is the cheapest and fastest way. Afterwards you have to extract your chosen Biobrick out of your bacteria. Secondly, when you completed your construct in form of a plasmid, you might need to bring it into your final bacteria cells to test if it works.
For amplifying DNA from the registry, we transformed chemocompetent Top10 cells by applying the following protocol:
DNA from Registry Plate: 10 ul water is added to the corresponding well containing the desired BioBrick DNA and DNA is solved by incubation for 15 min at room temperature. 5 ul of DNA solution are added to 50 ul of chemocompetent Top10 cells and incubated on ice for 20 min. Afterwards, a heat shock is performed by incubation the bacteria on 42°C/50 s. After another 2 min on ice, the bacteria are plated onto LB-Amp agar plates.
Inoculation of LB agar plates
...
Inoculation of liquid LB medium
...
Qiagen Miniprep
...
Restriction digest
...
SAP digest
...
Gel electrophoresis
...
Gel extraction
...
Qiagen nucleotide removal kit
...
Ligation digest
...
Oligo annealing
...
Colony PCR
...
Sequencing
...
X-Gal assay
...
ONPG assay
...
Use of the photometer
...
Use of the plate reader
...
