Team:SDU-Denmark/protocols

From 2010.igem.org

(Difference between revisions)
(SEM 1.1)
 
(33 intermediate revisions not shown)
Line 609: Line 609:
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>
-
5. Measure absorbance using UV-Vis spectrophotometer at 450 nm, was preformed on a …. From MEMPHYS<br><br>
+
5. Measure absorbance using UV-Vis spectrophotometer at 450 nm  
 +
<br><br>
</p>
</p>
Line 619: Line 620:
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek<br> <br>
3. Re-suspend 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>
-
5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, For this particular purpose, we use a C-18 column, and the eluents used are as follows: <br>
+
5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, For this particular purpose, we use a Poroshell 120 EC-C18 (4,6 x 150 mm 2,7 micron)column, and the eluents used are as follows: <br>
A-buffer: 100% methanol with 0,1% trifluoroacetic acid. <br>
A-buffer: 100% methanol with 0,1% trifluoroacetic acid. <br>
B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid. <br>
B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid. <br>
Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer. <br>
Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer. <br>
-
Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. <br>
+
Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. Usage of the HPLC and instruction on how to use it was kindly provided by [http://www.sdu.dk/Om_SDU/Institutter_centre/C_FLinT FLINT]<br>
<br>
<br>
</p>
</p>
Line 652: Line 653:
''Microscopy''<br>
''Microscopy''<br>
-
1. 5uL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
+
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
3. Samples are examined under the microscope.<br><br>
3. Samples are examined under the microscope.<br><br>
=== PS1.2 ===
=== PS1.2 ===
-
A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br><br>
+
A protocol for optimizing the motility of E.coli MG1655 to use for microscopy<br>
 +
This protocol is designed based on preceeding pilot studies<br>
''Materials:''<br>
''Materials:''<br>
• LB media<br>
• LB media<br>
-
• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O. indsæt ref.)<br>
+
• 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:''
Line 671: Line 673:
''Microscopy''<br>
''Microscopy''<br>
-
1. 5uL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
+
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.<br>
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.<br>
3. Samples are examined under the microscope.<br><br>
3. Samples are examined under the microscope.<br><br>
-
This protocol is a modified protocol based on the one mentioned in (indsæt kilde)<br><br>
+
 
Line 701: Line 703:
== Flagella staining ==
== Flagella staining ==
<br>
<br>
-
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>
+
The following protocols are based on knowledge recived from [[https://2010.igem.org/Team:SDU-Denmark/protocols#References 2]]<br>
===FS1.1===  
===FS1.1===  
<p style="text-align: justify;">
<p style="text-align: justify;">
Line 750: Line 752:
===FS1.2===
===FS1.2===
<p style="text-align: justify;">
<p style="text-align: justify;">
-
Day 1: Bacteria were platede on agar plates and incubated at 37 degrees overnight.  The staining solutions were prepared.<br><br>
+
Day 1: Bacteria were platede on agar plates and incubated at 37 degrees celcius overnight.  The staining solutions were prepared.<br><br>
Solution I: <br>
Solution I: <br>
Line 837: Line 839:
•    20mM acetosyringone (39.3 acetosyringone dissolved in 1mL DMSO and 9mL ddH2O)<br>
•    20mM acetosyringone (39.3 acetosyringone dissolved in 1mL DMSO and 9mL ddH2O)<br>
•    LB media<br>
•    LB media<br>
-
•    35ug/uL chloramphenicol<br>
+
•    35µg/mL chloramphenicol<br>
''Protocol:''<br>
''Protocol:''<br>
-
1.    A colony is inoculated in 5mL LB media with 35ug/uL 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>
-
2.    Parallel cultivations of 25mL LB media, 35ug/uL chloramphenicol and acetosyringone concentrations of 0mM (control), 100mM, 200mM and 400mM respectively.<br>  
+
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>
 +
== 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>
 +
[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>
</p>
</p>
</div>
</div>
<div id="rightcolumn">
<div id="rightcolumn">
-
Almost like cake recipes... although cake is infinitely more delicious than bacteria.
+
Almost like cake recipes... although cake is infinitely more delicious than bacteria. <br><br>
 +
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>
<br>
<br>
<br>
<br>
__TOC__
__TOC__
 +
<br>
 +
<div id="bonus">
 +
<br>
 +
<br>
 +
<br>
 +
[https://2010.igem.org/Team:SDU-Denmark/project/activities/commoninterest/diller 8===D]
 +
</div>
</div>
</div>

Latest revision as of 03:58, 28 October 2010