Team:SDU-Denmark/protocols

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(Protocols)
 
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3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant<br><br>
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5. Measure absorbance using UV-Vis spectrophotometer at 450 nm, was preformed on a …. From [http://www.memphys.sdu.dk/ MEMPHYS]<br><br>
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5. Measure absorbance using UV-Vis spectrophotometer at 450 nm  
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=== PS1.2 ===
=== PS1.2 ===
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A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br><br>
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A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br>
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This protocol is designed based on preceeding pilot studies<br>
''Materials:''<br>
''Materials:''<br>
• LB media<br>
• LB media<br>
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• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O. '''''indsæt ref.''''')<br>
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• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 1]]<br>
• 1mM retinal<br>
• 1mM retinal<br>
''Protocol:''
''Protocol:''
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3. Samples are examined under the microscope.<br><br>
3. Samples are examined under the microscope.<br><br>
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This protocol is a modified protocol based on the one mentioned in (indsæt kilde)<br><br>
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== Flagella staining ==
== Flagella staining ==
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The following protocols are based on knowledge recived from the article: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44660 ''Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells''] <br>
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The following protocols are based on knowledge recived from [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 2]]<br>
===FS1.1===  
===FS1.1===  
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''Protocol:''<br>
''Protocol:''<br>
1.    A colony is inoculated in 5mL LB media with 35µg/mL chloramphenicol and incubated over night at 37°C and 180rpm.<br>
1.    A colony is inoculated in 5mL LB media with 35µg/mL chloramphenicol and incubated over night at 37°C and 180rpm.<br>
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2.    Parallel cultivations of 25mL LB media, 35µg/mL chloramphenicol and acetosyringone concentrations of 0mM (control), 100mM, 200mM and 400mM respectively.<br>  
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2.    Parallel cultivations of 25mL LB media, 35µg/mL chloramphenicol and acetosyringone concentrations of 0uM (control), 100uM, 200uM and 400uM respectively.<br>  
3.    The cultures were incubated in a waterbath of 37°C and 180rpm<br>
3.    The cultures were incubated in a waterbath of 37°C and 180rpm<br>
4.    The optical density at 550nm (OD550) was measured every two hours and samples were freezed at -80°C and used for fluorescence measurements (excitation at 584nm, emmitation at 607nm)<br><br>
4.    The optical density at 550nm (OD550) was measured every two hours and samples were freezed at -80°C and used for fluorescence measurements (excitation at 584nm, emmitation at 607nm)<br><br>
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== References ==
== References ==
[1]Khan S, Amoyaw K, Spudich JL, Reid GP, Trentham DR,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1260487/pdf/biophysj00100-0082.pdf Bacterial chemoreceptor signaling probed by flash photorelease of a caged serine], Biophys J. 1992 Apr;62(1)<br>  
[1]Khan S, Amoyaw K, Spudich JL, Reid GP, Trentham DR,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1260487/pdf/biophysj00100-0082.pdf Bacterial chemoreceptor signaling probed by flash photorelease of a caged serine], Biophys J. 1992 Apr;62(1)<br>  
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[2]Harshey RM, Matsuyama T,[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC44660/pdf/pnas01140-0333.pdf Dimorphic transition in Escherichia coli and Salmonella typhimurium: Surface-induced differentiation into hyperflagellate swarmer cells],Proc Natl Acad Sci U S A. 1994 August 30; 91(18): 8631–8635<br>
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Almost like cake recipes... although cake is infinitely more delicious than bacteria.
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Almost like cake recipes... although cake is infinitely more delicious than bacteria. <br><br>
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Get a template for calculating your ligation concentrations <br><html><a href="https://static.igem.org/mediawiki/2010/4/40/Team_SDUTemplate_for_ligation.ZIP" style="color: #50CC38; text-decoratio: bold; font-size: 24px;">HERE</a></html>
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[https://2010.igem.org/Team:SDU-Denmark/project/activities/commoninterest/diller 8===D]
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Latest revision as of 03:58, 28 October 2010