Team:Aachen/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
(→Measurement of fluorescence)
m (→Protocols)
 
(49 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=
-
= 2D detection of IPTG and HSL =
+
In our experiments, we often re-used protocols for basic experimental steps. For a better overview, we split our protocols into four categories:
-
 
+
-
== Chip production ==
+
-
 
+
-
=== 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 at RT (21&nbsp;°C).
+
-
# discard the supernatant
+
-
# re-suspend the pellet in 1&nbsp;mL tempered (~21&nbsp;°C) LB-medium .
+
-
 
+
-
 
+
-
=== Agar preparation ===
+
-
 
+
-
# autoclave 50&nbsp;mL medium with 1.5&nbsp;%&nbsp;(w/v) agarose (has to be multiplied with the number of chips prepared).
+
-
# cool it down to 45&nbsp;°C in a water bath.
+
-
 
+
-
 
+
-
=== Chip preparation ===
+
-
 
+
-
# mix the cooled medium with the cells by inverting gently.
+
-
# pour it in the chip form, avoiding bubble formation (!).
+
-
# wait for approximately 20 min until the agar has solidified.
+
-
# cut out the chips with a scalpel.
+
-
# put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
+
-
# incubate two chips for 1&nbsp;h at 37&nbsp;°C prior to induction.
+
 +
* 2D Detection of IPTG & HSL
 +
* Culture Media
 +
* Molecular Biological Methods
 +
* Analytical Methods
 +
To access the protocols, please click on the respective category panel below:
<center>
<center>
-
{{Team:Aachen/Figure|Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png|title=Sensor-chip manufacturing|subtitle=XXX|width=900px}}
+
<html><ul class="team-grid" style="width:1064px;">
-
</center>
+
-
== Measurement of fluorescence ==
+
<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/detection" style="color:black;">
 +
    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <div class="menukachel" style="top: 32%;line-height: 1.5em;">2D Detection of<br/>IPTG & HSL</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/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
-
= Culture medium and culture conditions =
+
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
-
== Media ==
+
<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black;">
-
=== LB medium ===
+
    <div class="team-item team-info" style="width:214px;height:214px;" >
-
# weight components
+
      <div class="menukachel" style="top: 40%;line-height: 1.5em;">Culture Media</div>
-
#: '''5&nbsp;g/L NaCl'''
+
      <!-- <br/><br/>
-
#: '''10&nbsp;g/L tryptone'''
+
      <b>Principle of Operation</br>
-
#: '''5&nbsp;g/L yeast extract'''
+
      <br/><br/>
-
#: (15&nbsp;g/L agar for plates)
+
      click for more information -->
-
# fill up to 1&nbsp;L with deionized water
+
    </div>
-
# '''mix well''' by shaking
+
<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>
-
# autoclave
+
  </li>
-
## 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!)
+
 +
<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>
-
=== TB medium ===
+
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
-
# components 1:
+
<a href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black;">
-
#: '''4&nbsp;mL/L glycerol'''
+
    <div class="team-item team-info" style="width:214px;height:214px;" >
-
#: '''12&nbsp;g/L tryptone'''
+
      <div class="menukachel" style="top: 32%;line-height: 1.5em;">Analytical Methods</div>
-
#: '''24&nbsp;g/L yeast extract'''
+
      <!-- <br/><br/>
-
# fill up to 900&nbsp;mL with deionized water
+
      <b>Principle of Operation</br>
-
# '''mix well''' by shaking
+
      <br/><br/>
-
# autoclave
+
      click for more information -->
-
# components 2:
+
    </div>
-
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''
+
    <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>
-
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''
+
  </li>
-
# 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
+
-
 
+
</ul></html>
-
=== Hartmans minimal medium (HM) ===
+
-
 
+
-
=== M9 minimal medium (M9) ===
+
-
<center>
+
-
{| class="wikitable centered"
+
-
! '''Components for 1&nbsp;L''' !! '''Volume'''
+
-
|-
+
-
| bidest. water || style="text-align:right"| 778.667&nbsp;mL
+
-
|-
+
-
| 10x Salt solution || style="text-align:right"| 100&nbsp;mL
+
-
|-
+
-
| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;mL
+
-
|-
+
-
| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;mL
+
-
|-
+
-
| 1000x Trace elements || style="text-align:right"| 1&nbsp;mL
+
-
|-
+
-
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;mL
+
-
|}
+
-
 
+
-
 
+
-
{| class="wikitable centered"
+
-
| colspan="3"| '''10x Salt solution'''
+
-
|-
+
-
! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''
+
-
|-
+
-
| BisTris || style="text-align:right"| 95&nbsp;mM         || style="text-align:right"| 200,000&nbsp;mg/L
+
-
|-
+
-
| Ammmonium chloride || style="text-align:right"| 60&nbsp;mM || style="text-align:right"| 32,100&nbsp;mg/L
+
-
|-
+
-
| Sodium citrate || style="text-align:right"| 12.5&nbsp;mM || style="text-align:right"| 27,000&nbsp;mg/L
+
-
|-
+
-
| Monopotassium phosphate || style="text-align:right"| 3&nbsp;mM || style="text-align:right"| 4,170&nbsp;mg/L
+
-
|-
+
-
| Dipotassium phosphate || style="text-align:right"| 0.7&nbsp;mM || style="text-align:right"| 1,590&nbsp;mg/L
+
-
|-
+
-
|}
+
-
 
+
-
{| class="wikitable centered"
+
-
| colspan="3"| '''1000x Trace elements'''
+
-
|-
+
-
! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''
+
-
|-
+
-
| Iron(III) chloride  || style="text-align:right"| 50&nbsp;mM  || style="text-align:right"| 13,515&nbsp;mg/L
+
-
|- 
+
-
| Calcium chloride || style="text-align:right"| 20&nbsp;mM || style="text-align:right"| 2,220&nbsp;mg/L
+
-
|-
+
-
| Manganese(II) chloride      || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,258&nbsp;mg/L
+
-
|-
+
-
| Zinc sulfate || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,615&nbsp;mg/L
+
-
|-
+
-
| Cobalt(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 260&nbsp;mg/L
+
-
|-
+
-
| Copper(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 269&nbsp;mg/L
+
-
|-
+
-
| Nickel(II) chloride || style="text-align:right"| 2&nbsp;mM    || style="text-align:right"| 259&nbsp;mg/L
+
-
|-
+
-
| Sodium molybdate    || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 412&nbsp;mg/L
+
-
|-
+
-
| Sodium selenite || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 346&nbsp;mg/L
+
-
|-
+
-
| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L
+
-
|-
+
-
| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;mL
+
-
|-
+
-
|}
+
</center>
</center>
-
 
-
 
-
=== SOC ===
 
-
# components
 
-
#: '''0,5&nbsp;% yeast extract'''
 
-
#: '''2&nbsp;% tryptone'''
 
-
#: '''10&nbsp;mM NaCl'''
 
-
#: '''2.5&nbsp;mM KCl'''
 
-
#: '''20&nbsp;mM MgSO<sub>4</sub> '''
 
-
# fill up with deionized water
 
-
# adjust to pH 7.5 with NaOH
 
-
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)
 
-
 
-
= Genetic methods =
 
-
== Cloning ==
 
-
=== Restriction Digest ===
 
-
 
-
=== Ligation ===
 
-
 
-
=== Gibson Assembly ===
 
-
 
-
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].
 
-
 
-
# Set up the reaction according to the table below on ice (2-3 fragment assembly).
 
-
# 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.
 
-
# Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
 
-
 
-
<center>
 
-
{| class="wikitable" style="text-align: right;"
 
-
|-
 
-
| '''Total Amount of Fragments''' || 0.02-0.5&nbsp;pmols
 
-
|-
 
-
| '''Gibson Assembly Master Mix (2X)''' || 10&nbsp;µl
 
-
|-
 
-
| '''Deionized H<sub>2</sub>O''' || 10-X&nbsp;µl
 
-
|-
 
-
| '''Total Volume''' || '''20&nbsp;µl'''
 
-
|-
 
-
|}
 
-
</center>
 
-
 
-
== 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 ==
 
-
 
-
We have used several different types of PCR throughout our project:
 
-
 
-
* colony PCR
 
-
* check PCR
 
-
* gradient PCR
 
-
* SOE PCR
 
-
* touchdown PCR
 
-
* QuikChange(Ligation-During-Amplification)
 
-
 
-
The scope of appplication as well as the conduct are described below.
 
-
 
-
 
-
=== Colony PCR /Check 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
 
-
 
-
<center>
 
-
{| class="wikitable"
 
-
! parameter !! duration !! temp [°C] !!
 
-
|-
 
-
| denature||5:00||95 ||
 
-
|-
 
-
| '''anneal'''||00:30||56 || rowspan="3" | 30 cycles
 
-
|-
 
-
| '''elongate'''||01:00 per kb||72
 
-
|-
 
-
| '''denature'''||00:30||95
 
-
|-
 
-
| elongate||05:00||72 || rowspan="2" |
 
-
|-
 
-
| store||forever||8
 
-
|}
 
-
</center>
 
-
 
-
 
-
=== gradient PCR ===
 
-
 
-
=== SOE PCR ===
 
-
 
-
=== touchdown PCR ===
 
-
 
-
=== QuikChange ===
 
-
 
-
= Analytical methods =
 
-
 
-
== Agarose gel electrophoresis==
 
-
 
-
Separation of DNA or RNA
 
-
 
-
# take 5µl of the PCR product
 
-
# mix with 1µl loading dye
 
-
# apply onto agarose gel together with a marker
 
-
# run at 120°C for 40 minutes for a full gel
 
-
 
-
== SDS-PAGE ==
 
-
=== Cell preparation ===
 
-
* lysis cell pellet in lysis buffer
 
-
* centrifuge for 15&nbsp;min at 13.000 rpm
 
-
* mix the supernatant with 2x lammli buffer with β-mercaptoethanol
 
-
* denatured for 5&nbsp;min at 95&nbsp;°C
 
-
* sample to the gel
 
-
 
-
For some SDS-PAGEs, we used BioRad ready made gels.
 
-
 
-
The recipe of the self-made SDS is as follows:
 
-
 
-
=== 1.5x Buffer ===
 
-
* 1.5&nbsp;M Tris-Cl pH = 8.8
 
-
* in 1&nbsp;L is 40&nbsp;ml 10&nbsp;% SDS
 
-
 
-
=== Gels ===
 
-
<center>
 
-
{| class="wikitable" style="text-align: right;"
 
-
!
 
-
!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12 % 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'''
 
-
|-
 
-
| '''H{{sub|2}}O'''
 
-
| style="border-left: 2px solid #404040;"| 1.65&nbsp;mL || 3.3&nbsp;mL || 6.6&nbsp;mL
 
-
| style="border-left: 2px solid #404040;"| 1.5&nbsp;mL || 3&nbsp;mL || 6&nbsp;mL
 
-
|-
 
-
| '''1.5x Gel Buffer'''
 
-
| style="border-left: 2px solid #404040;"| 1.3&nbsp;mL || 2.6&nbsp;mL || 5.2&nbsp;mL
 
-
| style="border-left: 2px solid #404040;"| 0.65&nbsp;mL || 1.3&nbsp;mL || 2.6&nbsp;mL
 
-
|-
 
-
| '''30 % Acrylamide (37.5:1)'''
 
-
| style="border-left: 2px solid #404040;"| 2&nbsp;mL || 4&nbsp;mL || 8&nbsp;mL
 
-
| style="border-left: 2px solid #404040;"| 0.325&nbsp;mL || 0.65&nbsp;mL || 1.3&nbsp;mL
 
-
|-
 
-
| '''10 % APS'''
 
-
| style="border-left: 2px solid #404040;"| 50&nbsp;µL || 100&nbsp;µL || 200&nbsp;µL
 
-
| style="border-left: 2px solid #404040;"| 25&nbsp;µL || 50&nbsp;µL || 100&nbsp;µL
 
-
|-
 
-
| '''TEMED'''
 
-
| style="border-left: 2px solid #404040;"| 10&nbsp;µL || 20&nbsp;µL || 40&nbsp;µL
 
-
| style="border-left: 2px solid #404040;"| 5&nbsp;µL || 10&nbsp;µL || 20&nbsp;µL
 
-
|-
 
-
|}
 
-
</center>
 
-
 
-
==Run gel==
 
-
* apply the prepared samples together with a protein marker on the gel
 
-
* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V
 
-
 
-
== Bradford assay ==
 
-
 
-
Determination of protein concentration
 
-
 
-
== Measurement of fluorescence ==
 
-
 
-
The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
 
-
 
-
* volume of sample in each well: 100µl
 
-
* measure GFP fluorescence at an excitation wavelength of 496&nbsp;nm and an emission wavelength at 516&nbsp;nm
 
-
 
-
== Measurement of optical density ==
 
-
 
-
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:

Contact Disclaimer

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