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| + | __NOTOC__ |
| + | {{CSS/Main}} |
| + | {{Team:Aachen/Stylesheet}} |
| {{Team:Aachen/Header}} | | {{Team:Aachen/Header}} |
| + | <span id="partners"></span> |
| | | |
- | __TOC__
| + | =Protocols= |
| | | |
- | = Media =
| + | In our experiments, we often re-used protocols for basic experimental steps. For a better overview, we split our protocols into four categories: |
- | == LB medium ==
| + | |
- | # Weight components
| + | |
- | #: '''5 g/L NaCl'''
| + | |
- | #: '''10 g/L tryptone'''
| + | |
- | #: '''5 g/L yeast extract'''
| + | |
- | #: (15 g/L agar for plates)
| + | |
- | # Fill up to 1 L with deionized water
| + | |
- | # '''Mix well''' by shaking
| + | |
- | # Autoclave
| + | |
- | ## Autoclaving tape, caps slightly unscrewed
| + | |
- | ## Base of the pot has to be covered with deionized water
| + | |
- | ## Close lid
| + | |
- | ## Heat '''level 3 until the pressure valve opens'''
| + | |
- | ## Reduce '''heat level to 1.5'''
| + | |
- | ## Set timer to '''20 minutes'''
| + | |
- | ## Turn heater off
| + | |
- | ## '''Wait until the pressure valve retracts''' (30-45 minutes)
| + | |
- | ## Open, close caps & shake
| + | |
- | # For plates, wait until you can touch the bottle ('''<60 °C''', clean bench!)
| + | |
- | # Add antibiotics (1 µL/mL) and '''shake''' (gloves!)
| + | |
| | | |
- | == TB medium ==
| + | * 2D Detection of IPTG & HSL |
- | # Components 1:
| + | * Culture Media |
- | #: '''4 mL/L glycerol'''
| + | * Molecular Biological Methods |
- | #: '''12 g/L tryptone'''
| + | * Analytical Methods |
- | #: '''24 g/L yeast extract'''
| + | |
- | # Fill up to 900 mL with deionized water
| + | |
- | # '''Mix well''' by shaking
| + | |
- | # Autoclave
| + | |
- | # Components 2:
| + | |
- | #: '''0.17 M KH<sub>2</sub>PO<sub>4</sub>'''
| + | |
- | #: '''0.72 M K<sub>2</sub>HPO<sub>4</sub>'''
| + | |
- | # Dissolve in 100 mL deionized water and sterilize it by passing it through a filter
| + | |
- | # After autoclaving and cooling down, add sterile phosphate solutions
| + | |
| | | |
- | == Hartmans minimal medium (HM) == | + | To access the protocols, please click on the respective category panel below: |
| + | <center> |
| + | <html><ul class="team-grid" style="width:1064px;"> |
| | | |
- | == SOC == | + | <li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"> |
- | # Components
| + | <a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black;"> |
- | #: '''0,5 % yeast extract'''
| + | <div class="team-item team-info" style="width:214px;height:214px;" > |
- | #: '''2 % tryptone'''
| + | <div class="menukachel" style="top: 32%;line-height: 1.5em;">2D Detection of<br/>IPTG & HSL</div> |
- | #: '''10 mM NaCl'''
| + | <!-- <br/><br/> |
- | #: '''2.5 mM KCl'''
| + | <b>Principle of Operation</br> |
- | #: '''20 mM MgSO<sub>4</sub> '''
| + | <br/><br/> |
- | # Fill up with deionized water
| + | click for more information --> |
- | # Adjust to pH 7.5 with NaOH
| + | </div> |
- | # After autoclaving, add 20 mM sterile glucose solution (filter sterilization)
| + | <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> |
- | | + | </li> |
- | = Agar Chips =
| + | |
- | | + | |
- | '''Cell preparation'''
| + | |
- | | + | |
- | # Over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
| + | |
- | # Centrifuge all 50 mL by 3000 g for 10 min.
| + | |
- | # Discard the supernatant
| + | |
- | # Re-suspend the pellet in 1 mL medium at RT
| + | |
- | | + | |
- | | + | |
- | '''Agar preparation'''
| + | |
- | | + | |
- | # Autoclave 50 mL medium with 1.5 % agarose
| + | |
- | # Cool it down to 45 °C in a water bath
| + | |
- | | + | |
- | | + | |
- | '''Chip production'''
| + | |
- | | + | |
- | # Mix the cooled medium with the cells by inverting gently
| + | |
- | # Pour it in the chip form, avoiding bubble formation (!)
| + | |
- | # Wait for ca 20 min until the agar has solidified
| + | |
- | # Cut out the chips with a scalpel
| + | |
- | # Put ever two chips into a labeled petri dish
| + | |
- | # Incubate for 1 h at 37 °C
| + | |
- | | + | |
- | = Transformation = | + | |
- | == Heat Shock ==
| + | |
- | # Thaw cells on ice
| + | |
- | # Add 1 µL of plasmid DNA
| + | |
- | # Incubate on ice for 30 min
| + | |
- | # Heat shock at 42 °C for 60 s
| + | |
- | # Incubate on ice for 5 min
| + | |
- | # Add 200 µL of SOC media
| + | |
- | # Incubate at 37 °C for 2 h
| + | |
- | # Plate 20 and 200 µL on plates supplemented with the appropiate antibiotic
| + | |
- | | + | |
- | == Electroporation ==
| + | |
- | # add 1 μL plasmid to electrocompetent cells
| + | |
- | # put DNA/ cell suspension in electroporation cuvette
| + | |
- | # wipe dry the electroporator
| + | |
- | # use a small plastic pipette to place the cells
| + | |
- | # pulse: 2.5 kV, 200-400 Ω, 25 μF (for ''E.coli'')
| + | |
- | # immediatly add 1 mL LB and incubate for 2 h at 37 °C
| + | |
- | # plate 50 μL on selective medium plate
| + | |
- | # centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μL LB and plate it on selective medium plate
| + | |
- | | + | |
- | = PCR =
| + | |
- | == Colony PCR ==
| + | |
- | '''with GoTaq Mast Mix'''
| + | |
- | | + | |
- | * 12.5 µl GoTaq Master Mix
| + | |
- | * 1 µl primer_F
| + | |
- | * 1 µl primer_R
| + | |
- | * pick colony with tip and suspend in PCR tube
| + | |
- | * 9.5 µl ddH<sub>2</sub>O
| + | |
- | | + | |
- | {| class="wikitable"
| + | |
- | ! parameter !! duration !! temp [°C]
| + | |
- | |-
| + | |
- | | denature||5:00||95
| + | |
- | |-
| + | |
- | | '''anneal'''||00:30||56
| + | |
- | |-
| + | |
- | | '''elongate'''||01:00 per kb||72
| + | |
- | |-
| + | |
- | | '''denature'''||00:30||95
| + | |
- | |-
| + | |
- | | elongate||05:00||72
| + | |
- | |-
| + | |
- | | store||forever||8
| + | |
- | |}
| + | |
- | → 30 cycles
| + | |
- | | + | |
- | == QuikChange ==
| + | |
- | | + | |
- | = Clonings =
| + | |
- | == Restriction Digest ==
| + | |
- | | + | |
- | == Ligation ==
| + | |
- | | + | |
- | == Gibson Assembly ==
| + | |
- | | + | |
- | | + | |
- | | + | |
- | | + | |
- | = SDS-PAGE =
| + | |
- | For some SDS-PAGEs, we used BioRad ready made gels.
| + | |
- | | + | |
- | The recipe of the self-made SDS is as follows:
| + | |
- | | + | |
- | === 3.5x Buffer ===
| + | |
- | | + | |
- | | + | |
- | === Gels ===
| + | |
- | {| class="wikitable" style="text-align: right;"
| + | |
- | !
| + | |
- | !! style="border-left: 2px solid #404040;" colspan="3"|1 mm 12 % RUNNING Gel
| + | |
- | !! style="border-left: 2px solid #404040;" colspan="3"|1 mm 8 % RUNNING Gel
| + | |
- | !! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1 mm 4 % STACKING Gel
| + | |
- | |-
| + | |
- | |
| + | |
- | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
| + | |
- | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
| + | |
- | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
| + | |
- | |-
| + | |
- | | '''H{{sub|2}}O'''
| + | |
- | | style="border-left: 2px solid #404040;"| 1.669 mL || 3.337 mL || 6.674 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 2.45 mL || 4.9 mL || 9.8 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 1.421 mL || 2.842 mL || 5.684 mL
| + | |
- | |-
| + | |
- | | '''3.5x Gel Buffer'''
| + | |
- | | style="border-left: 2px solid #404040;"| 1.613 mL || 3.225 mL || 6.45 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 1.613 mL || 3.225 mL || 6.45 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 0.7 mL || 1.4 mL || 2.8 mL
| + | |
- | |-
| + | |
- | | '''30 % Acrylamide (37.5:1)'''
| + | |
- | | style="border-left: 2px solid #404040;"| 2.344 mL || 4.687 mL || 9.374 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 1.563 mL || 3.125 mL || 6.25 mL
| + | |
- | | style="border-left: 2px solid #404040;"| 0.329 mL || 0.658 mL || 1.316 mL
| + | |
- | |-
| + | |
- | | '''10 % APS'''
| + | |
- | | style="border-left: 2px solid #404040;"| 22.5 µL || 45 µL || 90 µL
| + | |
- | | style="border-left: 2px solid #404040;"| 22.5 µL || 45 µL || 90 µL
| + | |
- | | style="border-left: 2px solid #404040;"| 10.5 µL || 21 µL || 42 µL
| + | |
- | |-
| + | |
- | | '''TEMED'''
| + | |
- | | style="border-left: 2px solid #404040;"| 2.25 µL || 4.5 µL || 9 µL
| + | |
- | | style="border-left: 2px solid #404040;"| 2.25 µL || 4.5 µL || 9 µL
| + | |
- | | style="border-left: 2px solid #404040;"| 4.9 µL || 9.8 µL || 19.6 µL
| + | |
- | |-
| + | |
- | |}
| + | |
| | | |
| + | <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/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/> |
| + | click for more information --> |
| + | </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> |
| + | </li> |
| | | |
| + | <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;"> |
| + | <div class="menukachel" style="top: 25%;line-height: 1.5em;">Molecular Biological Methods</div> |
| + | <!-- <br/><br/> |
| + | <b>Principle of Operation</br> |
| + | <br/><br/> |
| + | click for more information --> |
| + | </div> |
| + | <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> |
| + | </li> |
| | | |
| + | <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;" > |
| + | <div class="menukachel" style="top: 32%;line-height: 1.5em;">Analytical Methods</div> |
| + | <!-- <br/><br/> |
| + | <b>Principle of Operation</br> |
| + | <br/><br/> |
| + | click for more information --> |
| + | </div> |
| + | <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}} |