Team:Aachen/Notebook/Protocols

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=Protocols=
=Protocols=
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<html><ul class="team-grid">
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In our experiments, we often re-used protocols for basic experimental steps. For a better overview, we split our protocols into four categories:
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<!-- Overview -->
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  <li><a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols" style="color:black">
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* 2D Detection of IPTG & HSL
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     <div class="team-item team-info" >
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* Culture Media
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* Molecular Biological Methods
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* Analytical Methods
 +
 
 +
To access the protocols, please click on the respective category panel below:
 +
<center>
 +
<html><ul class="team-grid" style="width:1064px;">
 +
 
 +
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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<a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black;">
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     <div class="team-item team-info" style="width:214px;height:214px;" >
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      <div class="menukachel" style="top: 32%;line-height: 1.5em;">2D Detection of<br/>IPTG & HSL</div>
 +
      <!-- <br/><br/>
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      <b>Principle of Operation</br>
       <br/><br/>
       <br/><br/>
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       <b> 2D detection of IPTG and HSL </b>
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       click for more information -->
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      <br/><br/>
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      Organization and preparation
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      <br/><br/>
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      <!-- click for more information -->
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     </div>
     </div>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
   </li>
   </li>
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  <li><a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols" style="color:black">
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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     <div class="team-item team-info" >
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<a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black;">
 +
     <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <div class="menukachel" style="top: 40%;line-height: 1.5em;">Culture Media</div>
 +
      <!-- <br/><br/>
 +
      <b>Principle of Operation</br>
       <br/><br/>
       <br/><br/>
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       <b> Culture medium and culture conditions </b>
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       click for more information -->
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      <br/><br/>
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      First work on BioBricks
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-
      <br/><br/>
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-
      <!-- click for more information -->
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     </div>
     </div>
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    <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/c/ca/Aachen_team_member_Florian_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a>
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<div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
   </li>
   </li>
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  <li><a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols" style="color:black">
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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     <div class="team-item team-info" >
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<a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black;">
 +
     <div class="team-item team-info" style="width:214px;height:214px;">
 +
      <div class="menukachel" style="top: 25%;line-height: 1.5em;">Molecular Biological Methods</div>
 +
      <!-- <br/><br/>
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      <b>Principle of Operation</br>
       <br/><br/>
       <br/><br/>
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       <b> Genetic methods </b>
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       click for more information -->        
-
      <br/><br/>
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      First creation of own BioBricks
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-
      <br/><br/>
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      <!-- click for more information -->
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     </div>
     </div>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
   </li>
   </li>
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  <li><a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols" style="color:black">
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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     <div class="team-item team-info" >
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<a href="http://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black;">
 +
     <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <div class="menukachel" style="top: 32%;line-height: 1.5em;">Analytical Methods</div>
 +
      <!-- <br/><br/>
 +
      <b>Principle of Operation</br>
       <br/><br/>
       <br/><br/>
-
       <b> Analytical methods </b>
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       click for more information -->
-
      <br/><br/>
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      Improving our chip technology
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-
      <br/><br/>
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-
      <!-- click for more information -->
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     </div>
     </div>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a>
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     <div class="team-item team-img" style="background: url(http://2014.igem.org/wiki/images/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
   </li>
   </li>
</ul></html>
</ul></html>
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{{Team:Aachen/BlockSeparator}}
 
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= 2D detection of IPTG and HSL =
 
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== Chip production ==
 
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=== Cell preparation ===
 
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# over night culture of sensor cells (50&nbsp;mL in a 250&nbsp;mL flask with) max. 16&nbsp;h
 
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# centrifuge all 50&nbsp;mL by 3000&nbsp;g for 10 min at RT (21&nbsp;°C).
 
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# discard the supernatant
 
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# re-suspend the pellet in 1&nbsp;mL tempered (~21&nbsp;°C) LB-medium .
 
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=== Agar preparation ===
 
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# autoclave 50&nbsp;mL medium with 1.5&nbsp;%&nbsp;(w/v) agarose (has to be multiplied with the number of chips prepared).
 
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# cool it down to 45&nbsp;°C in a water bath.
 
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=== Chip preparation ===
 
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# mix the cooled medium with the cells by inverting gently.
 
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# pour it in the chip form, avoiding bubble formation (!).
 
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# wait for approximately 20 min until the agar has solidified.
 
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# cut out the chips with a scalpel.
 
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# put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
 
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# incubate two chips for 1&nbsp;h at 37&nbsp;°C prior to induction.
 
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<center>
 
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{{Team:Aachen/Figure|Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png|title=Sensor-chip manufacturing|subtitle=XXX|width=900px}}
 
</center>
</center>
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== Measurement of fluorescence ==
 
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{{Team:Aachen/BlockSeparator}}
 
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= Culture medium and culture conditions =
 
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== Media ==
 
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=== LB medium ===
 
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# weight components
 
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#: '''5&nbsp;g/L NaCl'''
 
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#: '''10&nbsp;g/L tryptone'''
 
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#: '''5&nbsp;g/L yeast extract'''
 
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#: (15&nbsp;g/L agar for plates)
 
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# fill up to 1&nbsp;L with deionized water
 
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# '''mix well''' by shaking
 
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# autoclave
 
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## autoclaving tape, caps slightly unscrewed
 
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## base of the pot has to be covered with deionized water
 
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## close lid
 
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## heat '''level 3 until the pressure valve opens'''
 
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## reduce '''heat level to 1.5'''
 
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## set timer to '''20 minutes'''
 
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## turn heater off
 
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## '''wait until the pressure valve retracts''' (30-45 minutes)
 
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## open, close caps & shake
 
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# for plates, wait until you can touch the bottle ('''<60&nbsp;°C''', clean bench!)
 
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# add antibiotics (1&nbsp;µL/mL) and '''shake''' (gloves!)
 
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=== TB medium ===
 
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# components 1:
 
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#: '''4&nbsp;mL/L glycerol'''
 
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#: '''12&nbsp;g/L tryptone'''
 
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#: '''24&nbsp;g/L yeast extract'''
 
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# fill up to 900&nbsp;mL with deionized water
 
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# '''mix well''' by shaking
 
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# autoclave
 
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# components 2:
 
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#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
 
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#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
 
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# dissolve in 100&nbsp;mL deionized water and sterilize it by passing it through a filter
 
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# after autoclaving and cooling down, add sterile phosphate solutions
 
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=== Hartmans minimal medium (HM) ===
 
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=== M9 minimal medium (M9) ===
 
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<center>
 
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{| class="wikitable centered"
 
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! '''Components for 1&nbsp;L''' !! '''Volume'''
 
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|-
 
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| bidest. water || style="text-align:right"| 778.667&nbsp;mL
 
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|-
 
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| 10x Salt solution || style="text-align:right"| 100&nbsp;mL
 
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|-
 
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| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;mL
 
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|-
 
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| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;mL
 
-
|-
 
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| 1000x Trace elements || style="text-align:right"| 1&nbsp;mL
 
-
|-
 
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| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;mL
 
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|}
 
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{| class="wikitable centered"
 
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| colspan="3"| '''10x Salt solution'''
 
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|-
 
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! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''
 
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|-
 
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| BisTris || style="text-align:right"| 95&nbsp;mM         || style="text-align:right"| 200,000&nbsp;mg/L
 
-
|-
 
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| Ammmonium chloride || style="text-align:right"| 60&nbsp;mM || style="text-align:right"| 32,100&nbsp;mg/L
 
-
|-
 
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| Sodium citrate || style="text-align:right"| 12.5&nbsp;mM || style="text-align:right"| 27,000&nbsp;mg/L
 
-
|-
 
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| Monopotassium phosphate || style="text-align:right"| 3&nbsp;mM || style="text-align:right"| 4,170&nbsp;mg/L
 
-
|-
 
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| Dipotassium phosphate || style="text-align:right"| 0.7&nbsp;mM || style="text-align:right"| 1,590&nbsp;mg/L
 
-
|-
 
-
|}
 
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{| class="wikitable centered"
 
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| colspan="3"| '''1000x Trace elements'''
 
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|-
 
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! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''
 
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|-
 
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| Iron(III) chloride  || style="text-align:right"| 50&nbsp;mM  || style="text-align:right"| 13,515&nbsp;mg/L
 
-
|- 
 
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| Calcium chloride || style="text-align:right"| 20&nbsp;mM || style="text-align:right"| 2,220&nbsp;mg/L
 
-
|-
 
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| Manganese(II) chloride      || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,258&nbsp;mg/L
 
-
|-
 
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| Zinc sulfate || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,615&nbsp;mg/L
 
-
|-
 
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| Cobalt(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 260&nbsp;mg/L
 
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|-
 
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| Copper(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 269&nbsp;mg/L
 
-
|-
 
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| Nickel(II) chloride || style="text-align:right"| 2&nbsp;mM    || style="text-align:right"| 259&nbsp;mg/L
 
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|-
 
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| Sodium molybdate    || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 412&nbsp;mg/L
 
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|-
 
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| Sodium selenite || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 346&nbsp;mg/L
 
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|-
 
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| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L
 
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|-
 
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| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;mL
 
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|-
 
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|}
 
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</center>
 
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=== SOC ===
 
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# components
 
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#: '''0,5&nbsp;% yeast extract'''
 
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#: '''2&nbsp;% tryptone'''
 
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#: '''10&nbsp;mM NaCl'''
 
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#: '''2.5&nbsp;mM KCl'''
 
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#: '''20&nbsp;mM MgSO<sub>4</sub> '''
 
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# fill up with deionized water
 
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# adjust to pH 7.5 with NaOH
 
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# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
 
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{{Team:Aachen/BlockSeparator}}
 
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= Genetic methods =
 
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== Cloning ==
 
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=== Restriction Digest ===
 
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=== Ligation ===
 
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=== Gibson Assembly ===
 
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The Gibson Assembly was conducted according to the protocol published by [https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510 New England Biolabs].
 
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# Set up the reaction according to the table below on ice (2-3 fragment assembly).
 
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# Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
 
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# Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
 
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<center>
 
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{| class="wikitable" style="text-align: right;"
 
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|-
 
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| '''Total Amount of Fragments''' || 0.02-0.5&nbsp;pmols
 
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|-
 
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| '''Gibson Assembly Master Mix (2X)''' || 10&nbsp;µl
 
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|-
 
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| '''Deionized H<sub>2</sub>O''' || 10-X&nbsp;µl
 
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|-
 
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| '''Total Volume''' || '''20&nbsp;µl'''
 
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|-
 
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|}
 
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</center>
 
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== Transformation ==
 
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=== Heat Shock ===
 
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# thaw cells on ice
 
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# add 1&nbsp;µL of plasmid DNA
 
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# incubate on ice for 30 min
 
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# heat shock at 42&nbsp;°C for 60 s
 
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# incubate on ice for 5 min
 
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# add 200&nbsp;µL of SOC media
 
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# incubate at 37&nbsp;°C for 2&nbsp;h
 
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# plate 20&nbsp; and 200&nbsp;µL on plates supplemented with the appropiate antibiotic
 
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=== Electroporation ===
 
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# add 1&nbsp;μL plasmid to electrocompetent cells
 
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# put DNA/ cell suspension in electroporation cuvette
 
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# wipe dry the electroporator
 
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# use a small plastic pipette to place the cells
 
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# pulse: 2.5&nbsp;kV, 200-400&nbsp;Ω, 25&nbsp;μF (for ''E.coli'')
 
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# immediatly add 1&nbsp;mL LB and incubate for 2&nbsp;h at 37&nbsp;°C
 
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# plate 50&nbsp;μL on selective medium plate
 
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# centrifuge the rest (3000&nbsp;g, 20 min), discard supernatant, re-suspend the pellet in 50&nbsp;μL LB and plate it on selective medium plate
 
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== PCR ==
 
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We have used several different types of PCR throughout our project:
 
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* colony PCR
 
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* check PCR
 
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* gradient PCR
 
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* SOE PCR
 
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* touchdown PCR
 
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* QuikChange(Ligation-During-Amplification)
 
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The scope of appplication as well as the conduct are described below.
 
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=== Colony PCR /Check PCR ===
 
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'''With GoTaq Mast Mix'''
 
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* 12.5 µl GoTaq Master Mix
 
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* 1 µl primer_F
 
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* 1 µl primer_R
 
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* pick colony with tip and suspend in PCR tube
 
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* 9.5 µl ddH<sub>2</sub>O
 
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<center>
 
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{| class="wikitable"
 
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! parameter !! duration !! temp [°C] !!
 
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|-
 
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| denature||5:00||95 ||
 
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|-
 
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| '''anneal'''||00:30||56 || rowspan="3" | 30 cycles
 
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|-
 
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| '''elongate'''||01:00 per kb||72
 
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|-
 
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| '''denature'''||00:30||95
 
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|-
 
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| elongate||05:00||72 || rowspan="2" |
 
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|-
 
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| store||forever||8
 
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|}
 
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</center>
 
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=== gradient PCR ===
 
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=== SOE PCR ===
 
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=== touchdown PCR ===
 
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=== QuikChange ===
 
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{{Team:Aachen/BlockSeparator}}
 
-
 
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= Analytical methods =
 
-
 
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== Agarose gel electrophoresis==
 
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Separation of DNA or RNA
 
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# take 5µl of the PCR product
 
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# mix with 1µl loading dye
 
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# apply onto agarose gel together with a marker
 
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# run at 120°C for 40 minutes for a full gel
 
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== SDS-PAGE ==
 
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=== Cell preparation ===
 
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* lysis cell pellet in lysis buffer
 
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* centrifuge for 15&nbsp;min at 13.000 rpm
 
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* mix the supernatant with 2x lammli buffer with β-mercaptoethanol
 
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* denatured for 5&nbsp;min at 95&nbsp;°C
 
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* sample to the gel
 
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For some SDS-PAGEs, we used BioRad ready made gels.
 
-
 
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The recipe of the self-made SDS is as follows:
 
-
 
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=== 1.5x Buffer ===
 
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* 1.5&nbsp;M Tris-Cl pH = 8.8
 
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* in 1&nbsp;L is 40&nbsp;ml 10&nbsp;% SDS
 
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=== Gels ===
 
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<center>
 
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{| class="wikitable" style="text-align: right;"
 
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!
 
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!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12 % RUNNING Gel
 
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!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;mm 4 % STACKING Gel
 
-
|-
 
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|
 
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| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
 
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| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
 
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|-
 
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| '''H{{sub|2}}O'''
 
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| style="border-left: 2px solid #404040;"| 1.65&nbsp;mL || 3.3&nbsp;mL || 6.6&nbsp;mL
 
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| style="border-left: 2px solid #404040;"| 1.5&nbsp;mL || 3&nbsp;mL || 6&nbsp;mL
 
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|-
 
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| '''1.5x Gel Buffer'''
 
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| style="border-left: 2px solid #404040;"| 1.3&nbsp;mL || 2.6&nbsp;mL || 5.2&nbsp;mL
 
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| style="border-left: 2px solid #404040;"| 0.65&nbsp;mL || 1.3&nbsp;mL || 2.6&nbsp;mL
 
-
|-
 
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| '''30 % Acrylamide (37.5:1)'''
 
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| style="border-left: 2px solid #404040;"| 2&nbsp;mL || 4&nbsp;mL || 8&nbsp;mL
 
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| style="border-left: 2px solid #404040;"| 0.325&nbsp;mL || 0.65&nbsp;mL || 1.3&nbsp;mL
 
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|-
 
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| '''10 % APS'''
 
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| style="border-left: 2px solid #404040;"| 50&nbsp;µL || 100&nbsp;µL || 200&nbsp;µL
 
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| style="border-left: 2px solid #404040;"| 25&nbsp;µL || 50&nbsp;µL || 100&nbsp;µL
 
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|-
 
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| '''TEMED'''
 
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| style="border-left: 2px solid #404040;"| 10&nbsp;µL || 20&nbsp;µL || 40&nbsp;µL
 
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| style="border-left: 2px solid #404040;"| 5&nbsp;µL || 10&nbsp;µL || 20&nbsp;µL
 
-
|-
 
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|}
 
-
</center>
 
-
 
-
==Run gel==
 
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* apply the prepared samples together with a protein marker on the gel
 
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* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V
 
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== Bradford assay ==
 
-
 
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Determination of protein concentration
 
-
 
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== Measurement of fluorescence ==
 
-
 
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The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
 
-
 
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* volume of sample in each well: 100µl
 
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* measure GFP fluorescence at an excitation wavelength of 496&nbsp;nm and an emission wavelength at 516&nbsp;nm
 
-
 
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== Measurement of optical density ==
 
-
 
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Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.
 
-
 
{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Latest revision as of 00:49, 18 October 2014

Protocols

In our experiments, we often re-used protocols for basic experimental steps. For a better overview, we split our protocols into four categories:

  • 2D Detection of IPTG & HSL
  • Culture Media
  • Molecular Biological Methods
  • Analytical Methods

To access the protocols, please click on the respective category panel below: