Team:Aachen/Notebook/Protocols

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__NOTOC__
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{{CSS/Main}}
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{{Team:Aachen/Stylesheet}}
{{Team:Aachen/Header}}
{{Team:Aachen/Header}}
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<span id="partners"></span>
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__TOC__
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=Protocols=
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= Media =
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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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== 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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* 2D Detection of IPTG & HSL
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# components 1:
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* Culture Media
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#: '''4&nbsp;mL/L glycerol'''
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* Molecular Biological Methods
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#: '''12&nbsp;g/L tryptone'''
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* Analytical Methods
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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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To access the protocols, please click on the respective category panel below:
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<center>
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<html><ul class="team-grid" style="width:1064px;">
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== SOC ==
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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# components
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<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black;">
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#: '''0,5&nbsp;% yeast extract'''
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    <div class="team-item team-info" style="width:214px;height:214px;" >
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#: '''2&nbsp;% tryptone'''
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      <div class="menukachel" style="top: 32%;line-height: 1.5em;">2D Detection of<br/>IPTG & HSL</div>
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#: '''10&nbsp;mM NaCl'''
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      <!-- <br/><br/>
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#: '''2.5&nbsp;mM KCl'''
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      <b>Principle of Operation</br>
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#: '''20&nbsp;mM MgSO<sub>4</sub> '''
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      <br/><br/>
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# fill up with deionized water
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      click for more information -->
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# adjust to pH 7.5 with NaOH
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    </div>
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# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
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    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
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  </li>
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= Agar Chips =
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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.
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# discard the supernatant
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# re-suspend the pellet in 1&nbsp;mL medium at RT
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'''Agar preparation'''
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# autoclave 50&nbsp;mL medium with 1.5&nbsp;% agarose
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# cool it down to 45&nbsp;°C in a water bath
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'''Chip production'''
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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 ca 20 min until the agar has solidified
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# cut out the chips with a scalpel
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# put ever two chips into a labeled petri dish
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# incubate for 1&nbsp;h at 37&nbsp;°C
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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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== Colony 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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{| 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
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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
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|-
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| store||forever||8
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|}
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&rarr; 30 cycles
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== QuikChange ==
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= Clonings =
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== Restriction Digest ==
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== Ligation ==
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== Gibson Assembly ==
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= SDS-PAGE =
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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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=== 3.5x Buffer ===
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=== Gels ===
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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"|1&nbsp;mm 12 % RUNNING Gel
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!! style="border-left: 2px solid #404040;" colspan="3"|1&nbsp;mm 8 % 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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|
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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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| 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;"| 1.669&nbsp;mL || 3.337&nbsp;mL || 6.674&nbsp;mL
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| style="border-left: 2px solid #404040;"| 2.45&nbsp;mL || 4.9&nbsp;mL || 9.8&nbsp;mL
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| style="border-left: 2px solid #404040;"| 1.421&nbsp;mL || 2.842&nbsp;mL || 5.684&nbsp;mL
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|-
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| '''3.5x Gel Buffer'''
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| style="border-left: 2px solid #404040;"| 1.613&nbsp;mL || 3.225&nbsp;mL || 6.45&nbsp;mL
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| style="border-left: 2px solid #404040;"| 1.613&nbsp;mL || 3.225&nbsp;mL || 6.45&nbsp;mL
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| style="border-left: 2px solid #404040;"| 0.7&nbsp;mL || 1.4&nbsp;mL || 2.8&nbsp;mL
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|-
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| '''30 % Acrylamide (37.5:1)'''
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| style="border-left: 2px solid #404040;"| 2.344&nbsp;mL || 4.687&nbsp;mL || 9.374&nbsp;mL
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| style="border-left: 2px solid #404040;"| 1.563&nbsp;mL || 3.125&nbsp;mL || 6.25&nbsp;mL
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| style="border-left: 2px solid #404040;"| 0.329&nbsp;mL || 0.658&nbsp;mL || 1.316&nbsp;mL
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|-
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| '''10 % APS'''
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| style="border-left: 2px solid #404040;"| 22.5&nbsp;µL || 45&nbsp;µL || 90&nbsp;µL
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| style="border-left: 2px solid #404040;"| 22.5&nbsp;µL || 45&nbsp;µL || 90&nbsp;µL
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| style="border-left: 2px solid #404040;"| 10.5&nbsp;µL || 21&nbsp;µL || 42&nbsp;µL
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|-
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| '''TEMED'''
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| style="border-left: 2px solid #404040;"| 2.25&nbsp;µL || 4.5&nbsp;µL || 9&nbsp;µL
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| style="border-left: 2px solid #404040;"| 2.25&nbsp;µL || 4.5&nbsp;µL || 9&nbsp;µL
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| style="border-left: 2px solid #404040;"| 4.9&nbsp;µL || 9.8&nbsp;µL || 19.6&nbsp;µL
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|-
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|}
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" 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: 40%;line-height: 1.5em;">Culture Media</div>
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      <!-- <br/><br/>
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      <b>Principle of Operation</br>
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      <br/><br/>
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      click for more information -->
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    </div>
 +
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
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  </li>
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black;">
 +
    <div class="team-item team-info" style="width:214px;height:214px;">
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      <div class="menukachel" style="top: 25%;line-height: 1.5em;">Molecular Biological Methods</div>
 +
      <!-- <br/><br/>
 +
      <b>Principle of Operation</br>
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      <br/><br/>
 +
      click for more information -->       
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    </div>
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    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/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>
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  </li>
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black;">
 +
    <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;">Analytical Methods</div>
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      <!-- <br/><br/>
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      <b>Principle of Operation</br>
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      <br/><br/>
 +
      click for more information -->
 +
    </div>
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    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
 +
</ul></html>
 +
</center>
{{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:

Contact Disclaimer

Retrieved from "http://2014.igem.org/Team:Aachen/Notebook/Protocols"