Team:Braunschweig/Protocols

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<p><h1>Protocols</h1></p>
<p><h1>Protocols</h1></p>
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<p><img alt="Braunschweig Labbook" src="https://static.igem.org/mediawiki/2013/a/a6/Braunschweig_Reagenzgl%C3%A4ser.png" align="right" width="100" vspace="10" hspace="20" style="margin-right:5px" style="margin-top:10px"/>
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<p>In this section you will find detailed protocols of experimental procedures.<br></p>
<p>In this section you will find detailed protocols of experimental procedures.<br></p>
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<div class="protocols">
<div class="protocols">
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     <h2><a href="#Growthcurve">Measurement of Growth Curves</a></h2>
     <h2><a href="#Growthcurve">Measurement of Growth Curves</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures in 20 mL 2xYT medium containing chloramphenicol were inoculated directly from -80°C glycerol cell stock. Cells were grown in non-baffled-flasks at 37°C and 250 rpm.<br>  
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures in 20 mL 2xYT medium containing chloramphenicol were inoculated directly from -80°C glycerol cell stock. Cells were grown in non-baffled-flasks at 37°C and 250 rpm.<br>  
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The next day, main cultures in 75 mL 2xYT medium containing ampicillin were inoculated from overnight cultures to an OD520=0.5. Growth of each strain was observed with and without induction of the promoters regulation the ampicillin resistance gene. All cultures and measurements were conducted as duplicates.<br>  
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The next day, main cultures in 75 mL 2xYT medium containing ampicillin were inoculated from overnight cultures to an OD<sub>520</sub>=0.5. Growth of each strain was observed with and without induction of the promoters regulation the ampicillin resistance gene. All cultures and measurements were conducted as duplicates.<br>  
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For the induction of beta-lactamase (ampR) expression by P<sub>Rhl</sub> und P<sub>Las</sub> autoinducers N-buturyl-homoserine lactone and N-3-oxododecanoyl homoserine lactone were added respectively to a final concentration of 10 µmol/L. Cells were grown in non-baffled flasks at 37°C and 250 rpm. <br>  
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For the induction of beta-lactamase (<i>ampR</i>) expression by Prhl und Plas autoinducers N-buturyl-homoserine lactone and N-3-oxododecanoyl homoserine lactone were added respectively to a final concentration of 10 µmol/L. Cells were grown in non-baffled flasks at 37°C and 250 rpm. <br>  
Samples from each flask were taken at appropriate time points depending on the growth phase of the cells. Optical density was measured at a wavelength of 520 nm in order to avoid/minimize absorption by the chromoproteins (see absorption spectra). Cells were left to grow until the stationary phase was reached.
Samples from each flask were taken at appropriate time points depending on the growth phase of the cells. Optical density was measured at a wavelength of 520 nm in order to avoid/minimize absorption by the chromoproteins (see absorption spectra). Cells were left to grow until the stationary phase was reached.
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
<b>Chemically competent cells</b><br>
<b>Chemically competent cells</b><br>
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100 mL of 2xYT Media containing required antibiotic were inoculated and incubate at 37°C and 250 rpm until OD<sub>600</sub> = 0.5. Subsequently the cells were incubated on ice for 15 minutes and then centrifuged at 4 °C and 400 rpm for 5 minutes. The excess was discarded and the pellets resuspended in 7.5 mL ice cold TFB1. Afterwards the solution is incubated on ice for 90 minutes and then centrifuged for 5 minutes at 4000 rpm. Finally the pellets were resuspended, aliquoted and quick-frozen in liquid nitrogen. The vials were stored at -80°C.<br><br>
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100 mL of 2xYT media containing required antibiotic were inoculated and incubate at 37°C and 250 rpm until OD<sub>600</sub> = 0.5. Subsequently the cells were incubated on ice for 15 minutes and then centrifuged at 4 °C and 400 rpm for 5 minutes. The excess was discarded and the pellets resuspended in 7.5 mL ice cold TFB1. Afterwards the solution is incubated on ice for 90 minutes and then centrifuged for 5 minutes at 4000 rpm. Finally the pellets were resuspended, aliquoted and quick-frozen in liquid nitrogen. The vials were stored at -80°C.<br><br>
<b>Electrocompetent cells</b><br>
<b>Electrocompetent cells</b><br>
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Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.<br>  
Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.<br>  
First 2 mL of the cell suspension were spun down for 10 minutes (5000g).<br>  
First 2 mL of the cell suspension were spun down for 10 minutes (5000g).<br>  
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For the alkaline lysis of the DNA the pellet was resuspended in 250 μL solution I (RNAseA). 250 μL of solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 g for 10 minutes.<br>
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For the alkaline lysis of the DNA the pellet was resuspended in 250 μL solution I (RNAseA). 250 μL of solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000xg for 10 minutes.<br>
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Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000g). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10.000g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000g) and dried.<br>
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Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000xg). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10000xg). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000xg) and dried.<br>
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At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000g).  
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At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000xg).  
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<div id="Fluorescenceimaging" class="menuSection">
<div id="Fluorescenceimaging" class="menuSection">
     <h2><a href="#Fluorescenceimaging">Imaging of Cells producing Chromoproteins</a></h2>
     <h2><a href="#Fluorescenceimaging">Imaging of Cells producing Chromoproteins</a></h2>
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM N-buturyl homoserine lactone (P<sub>Rhl</sub>) or N-3-oxododecanoyl homoserine lactone (P<sub>Las</sub>) at 37°C and 250 rpm over night. The medium was inoculated directly from -80°C glycerol cell stock.  
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM N-buturyl homoserine lactone (Prhl) or N-3-oxododecanoyl homoserine lactone (Plas) at 37°C and 250 rpm over night. The medium was inoculated directly from -80°C glycerol cell stock.  
Cells were diluted with fresh 2xYT medium containing ampicillin and suspension was placed between to agar layers containing ampicllin as well.<br>  
Cells were diluted with fresh 2xYT medium containing ampicillin and suspension was placed between to agar layers containing ampicllin as well.<br>  
Cell growth and division were observed for 3 hours by exciting chromoproteins every five minutes at specific wavelength (see absorption spectra) using YFP und mCherry filters.  
Cell growth and division were observed for 3 hours by exciting chromoproteins every five minutes at specific wavelength (see absorption spectra) using YFP und mCherry filters.  
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<h1>Our sponsors</h1>
<h1>Our sponsors</h1>
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Latest revision as of 13:16, 27 October 2013

Protocols

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Braunschweig Labbook

In this section you will find detailed protocols of experimental procedures.

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Our sponsors

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