Team:Aachen/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
(→Colony PCR)
m (→Protocols)
 
(163 intermediate revisions not shown)
Line 1: Line 1:
 +
__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&nbsp;g/L NaCl'''
+
-
#: '''10&nbsp;g/L tryptone'''
+
-
#: '''5&nbsp;g/L yeast extract'''
+
-
#: (15&nbsp;g/L agar for plates)
+
-
# fill up to 1&nbsp;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&nbsp;°C''', clean bench!)
+
-
# add antibiotics (1&nbsp;µL/mL) and '''shake''' (gloves!)
+
-
== TB medium ==
+
* 2D Detection of IPTG & HSL
-
# components 1:
+
* Culture Media
-
#: '''4&nbsp;mL/L glycerol'''
+
* Molecular Biological Methods
-
#: '''12&nbsp;g/L tryptone'''
+
* Analytical Methods
-
#: '''24&nbsp;g/L yeast extract'''
+
-
# fill up to 900&nbsp;mL with deionized water
+
-
# '''mix well''' by shaking
+
-
# autoclave
+
-
# components 2:
+
-
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
+
-
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
+
-
# dissolve in 100&nbsp;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&nbsp;% yeast extract'''
+
    <div class="team-item team-info" style="width:214px;height:214px;" >
-
#: '''2&nbsp;% tryptone'''
+
      <div class="menukachel" style="top: 32%;line-height: 1.5em;">2D Detection of<br/>IPTG & HSL</div>
-
#: '''10&nbsp;mM NaCl'''
+
      <!-- <br/><br/>
-
#: '''2.5&nbsp;mM KCl'''
+
      <b>Principle of Operation</br>
-
#: '''20&nbsp;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&nbsp;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&nbsp;mL in a 250&nbsp;mL flask with) max. 16&nbsp;h
+
-
# centrifuge all 50&nbsp;mL by 3000&nbsp;g for 10 min.
+
-
# discard the supernatant
+
-
# re-suspend the pellet in 1&nbsp;mL medium at RT
+
-
 
+
-
 
+
-
'''Agar preparation'''
+
-
 
+
-
# autoclave 50&nbsp;mL medium with 1.5&nbsp;% agarose
+
-
# cool it down to 45&nbsp;°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&nbsp;h at 37&nbsp;°C
+
-
 
+
-
= Transformation =
+
-
== Heat Shock ==
+
-
# thaw cells on ice
+
-
# add 1&nbsp;µL of plasmid DNA
+
-
# incubate on ice for 30 min
+
-
# heat shock at 42&nbsp;°C for 60 s
+
-
# incubate on ice for 5 min
+
-
# add 200&nbsp;µL of SOC media
+
-
# incubate at 37&nbsp;°C for 2&nbsp;h
+
-
# plate 20&nbsp; and 200&nbsp;µL on plates supplemented with the appropiate antibiotic
+
-
 
+
-
== Electroporation ==
+
-
# add 1&nbsp;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&nbsp;kV, 200-400&nbsp;Ω, 25&nbsp;μF (for ''E.coli'')
+
-
# immediatly add 1&nbsp;mL LB and incubate for 2&nbsp;h at 37&nbsp;°C
+
-
# plate 50&nbsp;μL on selective medium plate
+
-
# 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
+
-
 
+
-
= 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
+
-
|}
+
-
 
+
-
== 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&nbsp;mm 12 % RUNNING Gel
+
-
!! style="border-left: 2px solid #404040;" colspan="3"|1&nbsp;mm 8 % RUNNING Gel
+
-
!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;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&nbsp;mL || 3.337&nbsp;mL || 6.674&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 2.45&nbsp;mL || 4.9&nbsp;mL || 9.8&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 1.421&nbsp;mL || 2.842&nbsp;mL || 5.684&nbsp;mL
+
-
|-
+
-
| '''3.5x Gel Buffer'''
+
-
| style="border-left: 2px solid #404040;"| 1.613&nbsp;mL || 3.225&nbsp;mL || 6.45&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 1.613&nbsp;mL || 3.225&nbsp;mL || 6.45&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 0.7&nbsp;mL || 1.4&nbsp;mL || 2.8&nbsp;mL
+
-
|-
+
-
| '''30 % Acrylamide (37.5:1)'''
+
-
| style="border-left: 2px solid #404040;"| 2.344&nbsp;mL || 4.687&nbsp;mL || 9.374&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 1.563&nbsp;mL || 3.125&nbsp;mL || 6.25&nbsp;mL
+
-
| style="border-left: 2px solid #404040;"| 0.329&nbsp;mL || 0.658&nbsp;mL || 1.316&nbsp;mL
+
-
|-
+
-
| '''10 % APS'''
+
-
| style="border-left: 2px solid #404040;"| 22.5&nbsp;µL || 45&nbsp;µL || 90&nbsp;µL
+
-
| style="border-left: 2px solid #404040;"| 22.5&nbsp;µL || 45&nbsp;µL || 90&nbsp;µL
+
-
| style="border-left: 2px solid #404040;"| 10.5&nbsp;µL || 21&nbsp;µL || 42&nbsp;µL
+
-
|-
+
-
| '''TEMED'''
+
-
| style="border-left: 2px solid #404040;"| 2.25&nbsp;µL || 4.5&nbsp;µL || 9&nbsp;µL
+
-
| style="border-left: 2px solid #404040;"| 2.25&nbsp;µL || 4.5&nbsp;µL || 9&nbsp;µL
+
-
| style="border-left: 2px solid #404040;"| 4.9&nbsp;µL || 9.8&nbsp;µL || 19.6&nbsp;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}}

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"