Team:Uppsala/InterlabStudy
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- | document.getElementById("tab3").innerHTML = '<p><h2>Section I: Provenance & Release</h2><br> | + | document.getElementById("tab3").innerHTML = '<p><h2>Section I: Provenance & Release</h2><br><p><b>Measurements taken by:</b>Stephanie Herman, Martin Friberg and Nils Anlind<br><b>Construction of parts:</b> Martin Friberg<br><b>Acknowledgements:</b> Erik Gullberg for support operating FACS and provided reagents, team Uppsala iGEM 2014 for valuable discussion and support.</p><br><br><b>Dates of protocols:</b><br><br>Overnight Cultures: 2014-09-17 and 2014-09-25<br>FACS: 2014-09-18 and 2014-09-26<br><br><b>Inclusion of data in publication:</b><br>Nils Anlind - Yes<br>Martin Friberg - Yes<br>Stephanie Herman - Yes</p> <h2>Section II: Protocols</h2><p>Preparation of FACS-samples: Four colonies of each construct(taken from a re-streaked colony) were grown overnight in LB(about 6mL) for 18 hours in 37 degrees celsius with 300 rpm shaking. 2,5 µl culture was put into 500 µl PBS in a FACS-tube and was left to incubate for at least 1 hour in room temperature. <b>Manufacturer and model:</b><br>BD FACSAria IIu<br><br><b>Configurations:</b><br>FITC filter band pass 530/30, top 530 nm, 30 nm width, 480 nm blue laser. Voltage applied over the photomultiplier tube: 400 V.<br><br><b>Protocol to take measurements:</b><br>The sample tubes were loaded into the FACS and measured one at a time using BD FACSDiva™ software!<br><br> <b>Method to include or exclude each sample:</b><br><br>All samples measured was included. Individual cells that lacked fluorescence of GFP or BFP was exclude in the samples via setting a fluorescence threshold. In the cases of clear peak, the threshold was set around the peak. When there was an unclear peak, the threshold was adjusted via a negative control with no fluorescence so 1% of the negative control was included in the region.<br><br><b>Controls used:</b><br>Sterile filtered water sample was used to ensure low noise, and DH5-alpha cells without plasmids was used as non-fluorescent cell control.<br><br><b>What quantities were measured:</b><br>Fluorescence per cell.<br><br><b>Time for each set of measurements:</b> for one construct 5min (four measurements)<br><b>Cost of each set of measurements:</b> for one construct, about 5 USD.<br><b>Practical limits of quantity of samples:</b> ~24 constructs per session. The machine have a variating noise background that changes if the machine needs to be re-started. This leads to increased noise in some cases high enough that you cannot read low levels of expression.</p><h2>Section III: Measured quantities</h2><p><b>Units:</b> Fluorescence measured in AU (arbitrary units) per cell.<br><b>Equivalent in SI-units:</b> No equivalent in SI-units since measurements in absolute units was not possible. Output data depends on voltage applied over the photomultiplier tube.<br><br><b>Range of measurement:</b> Since the device measures single cells the size of the sample is only limited by the runtime, the device measures 2*104 cells/s. For this study 105 cells/sample were measured.<br><b>Significant numbers on measurement:</b> Depends upon biological variation which is hard to determine.<br><b>Precision the same in the entire range? if not, how does it differ:</b> Precision is higher at higher fluorescence since the influence of background noise is decreased.<br><b>How did you answer questions above?</b> With the help of our advisor, Erik Gullberg.<br><br><b>Instrument last calibrated:</b> 2014-09-15<br><b>How was it calibrated:</b> Software calibration using BD Cytometer Setup & Tracking Beads (#642412)</p><h2>Section IV: Measurements</h2> <p><b>See attached file:</b> FACS_ILS.<br>Sheet “Sample data” contain the raw data obtained from measurements.<br> Sheet “Construct data” contains summarized data for each construct. Geometrical mean and standard deviation of the four samples was calculated and then normalized after J23101 expression during the same FACS-run to get it in relative promoter strength.</p> <a href="https://static.igem.org/mediawiki/2014/7/78/Uppsala-igem2014-ILS_Raw_Data.xls">Interlab study raw data</a>'; |
- | <p><b>Measurements taken by:</b>Stephanie Herman, Martin Friberg and Nils Anlind<br><b>Construction of parts:</b> Martin Friberg<br><b>Acknowledgements:</b> Erik Gullberg for support operating FACS and provided reagents, team Uppsala iGEM 2014 for valuable discussion and support.</p><br><br><b>Dates of protocols:</b><br><br>Overnight Cultures: 2014-09-17 and 2014-09-25<br> | + | |
- | FACS: 2014-09-18 and 2014-09-26<br><br><b>Inclusion of data in publication:</b><br>Nils Anlind - Yes<br>Martin Friberg - Yes<br>Stephanie Herman - Yes</p> <h2>Section II: Protocols</h2><p>Preparation of FACS-samples: Four colonies of each construct(taken from a re-streaked colony) were grown overnight in LB(about 6mL) for 18 hours in 37 degrees celsius with 300 rpm shaking. 2,5 µl culture was put into 500 µl PBS in a FACS-tube and was left to incubate for at least 1 hour in room temperature. <b>Manufacturer and model:</b><br>BD FACSAria IIu<br><br><b>Configurations:</b><br>FITC filter band pass 530/30, top 530 nm, 30 nm width, 480 nm blue laser. Voltage applied over the photomultiplier tube: 400 V.<br><br><b>Protocol to take measurements:</b><br>The sample tubes were loaded into the FACS and measured one at a time using BD FACSDiva™ software!<br><br> <b>Method to include or exclude each sample:</b><br><br>All samples measured was included. Individual cells that lacked fluorescence of GFP or BFP was exclude in the samples via setting a fluorescence threshold. In the cases of clear peak, the threshold was set around the peak. When there was an unclear peak, the threshold was adjusted via a negative control with no fluorescence so 1% of the negative control was included in the region.<br><br><b>Controls used:</b><br>Sterile filtered water sample was used to ensure low noise, and DH5-alpha cells without plasmids was used as non-fluorescent cell control.<br><br><b>What quantities were measured:</b><br>Fluorescence per cell.<br><br><b>Time for each set of measurements:</b> for one construct 5min (four measurements)<br> | + | |
- | <b>Cost of each set of measurements:</b> for one construct, about 5 USD.<br><b>Practical limits of quantity of samples:</b> ~24 constructs per session. The machine have a variating noise background that changes if the machine needs to be re-started. This leads to increased noise in some cases high enough that you cannot read low levels of expression.</p><h2>Section III: Measured quantities</h2><p><b>Units:</b> Fluorescence measured in AU (arbitrary units) per cell.<br><b>Equivalent in SI-units:</b> No equivalent in SI-units since measurements in absolute units was not possible. Output data depends on voltage applied over the photomultiplier tube.<br><br><b>Range of measurement:</b> Since the device measures single cells the size of the sample is only limited by the runtime, the device measures 2*104 cells/s. For this study 105 cells/sample were measured.<br><b>Significant numbers on measurement:</b> Depends upon biological variation which is hard to determine.<br><b>Precision the same in the entire range? if not, how does it differ:</b> Precision is higher at higher fluorescence since the influence of background noise is decreased.<br><b>How did you answer questions above?</b> With the help of our advisor, Erik Gullberg.<br><br><b>Instrument last calibrated:</b> 2014-09-15<br><b>How was it calibrated:</b> Software calibration using BD Cytometer Setup & Tracking Beads (#642412)</p><h2>Section IV: Measurements</h2> <p><b>See attached file:</b> FACS_ILS.<br>Sheet “Sample data” contain the raw data obtained from measurements.<br> Sheet “Construct data” contains summarized data for each construct. Geometrical mean and standard deviation of the four samples was calculated and then normalized after J23101 expression during the same FACS-run to get it in relative promoter strength.</p> <a href="https://static.igem.org/mediawiki/2014/7/78/Uppsala-igem2014-ILS_Raw_Data.xls">Interlab study raw data</a>'; | + | |
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Revision as of 12:57, 15 October 2014
Stephanie Herman
Teresa Reinli
Joakim Hellner
Alexander Virtanen
Jennifer Rosenius
Marcus Hong
Miranda Stiernborg
Tim Hagelby Edström
Viktor Blomkvist
Megha Biradar
Niklas Handin
Jonas Mattisson
Arina Gromov
Nils Anlind
Eric Sandström
Gunta Celma
Oliver Possnert
Martin Friberg
Kira Karlsson
Christoffer Andersson
Laura Pacoste
Andries Willem Boers
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