Difference between revisions of "Team:ETH Zurich/Materials"

Line 20: Line 20:
 
<p>Cell line derived from acute myeloid leukemia. We used this cell line as a model line for cancer cells with elevated lactate production and TRAIL susceptibility (papers).</p>
 
<p>Cell line derived from acute myeloid leukemia. We used this cell line as a model line for cancer cells with elevated lactate production and TRAIL susceptibility (papers).</p>
  
 +
<h3>SK-BR-3</h3>
 +
 +
<p>HER2-positive breast cancer cell line. We used this cell line to validate the chimeric receptors of the <a href="https://2015.igem.org/Team:Stockholm">Stockholm</a> iGEM team.</p>
  
 
<h3>3T3</h3>
 
<h3>3T3</h3>
  
 
<p>Murine fibroblast cell line. We used this cell line as a model line for healthy cells which are restistant to TRAIL and produce normal levels of lactate.</p>  
 
<p>Murine fibroblast cell line. We used this cell line as a model line for healthy cells which are restistant to TRAIL and produce normal levels of lactate.</p>  
−
 
−
<h3>SK-BR-3</h3>
 
−
 
−
<p>HER2-positive breast cancer cell line. We used this cell line to validate the chimeric receptors of the <a href="https://2015.igem.org/Team:Stockholm">Stockholm</a> iGEM team.</p>
 
  
 
</div>
 
</div>
Line 36: Line 35:
  
 
<h2>Machines</h2>
 
<h2>Machines</h2>
−
 
−
 
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Nano Drop</h3>
+
<h3>Absorbance Measurments</h3>
 
<ul>
 
<ul>
−
<li>Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c Software</li>
+
<li>Novaspec Plus™ Visible Spectrophotometer</li>
−
<a href="http://www.nanodrop.com/Productnd2000overview.aspx"><p>for further information click here</p></a>
+
<a href="http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11012&catalogId=10101&productId=20430&storeId=11782&langId=-1"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:NanoDrop.JPG|center|thumb]]
+
[[File:ODPhotometer.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Plate Reader</h3>
+
<h3>Balance</h3>
 
<ul>
 
<ul>
−
<li>Tecan Infinite M200 Pro™ combined with Tecan iControl TM software</li>
+
<li>MettlerToledo XS4002S DeltaRange® Balance</li>
−
<a href="http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1"><p>for further information (platereader) click here</p></a>
+
<a href="http://us.mt.com/us/en/home/products/Laboratory_Weighing_Solutions/Precision_Balances/XS_Precision_Balances/XS_4002_S_Dual_Range.html"><p>for further information click here</p></a>
−
<a href="http://www.tecan.ch/platform/apps/product/index.asp?MenuID=1817&ID=1924&Menu=1&Item=21.7.8"><p>for further information (software) click here</p></a>
+
 
</html>
 
</html>
−
[[File:TecanPlateReader.JPG|center|thumb]]
+
[[File:Balance.JPG|center|thumb]]
−
<html>
+
−
</html>
+
−
[[File:ProgramTecanReader.JPG|center|thumb]]
+
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Absorbance Measurments</h3>
+
<h3>Gel Electrophoresis</h3>
 
<ul>
 
<ul>
−
<li>Novaspec Plus™ Visible Spectrophotometer</li>
+
<li>Helixx Technologies MyRun®</li>
−
<a href="http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11012&catalogId=10101&productId=20430&storeId=11782&langId=-1"><p>for further information click here</p></a>
+
<a href="http://www.helixxtec.com/helixx/c/iMyRun/iMyRun.htm"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:ODPhotometer.JPG|center|thumb]]
+
[[File:GelRunningThing.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>UV- and Dark Hood</h3>
+
<h3>Emulsifier</h3>
 
<ul>
 
<ul>
−
<li>Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence</li>
+
<li>EmulsiFlex-C3 from AVESTIN, Inc. and AVESTIN Europe GmbH/li>
−
<a href="http://www.biostep.de/i18n_en_DE/products/Bio_Imaging_components_1776/Tripod__dark_hoods_and_accessories_1863/Dark_hoods_1865/Dark_Hood_DH_40_with_control_panel_and_display_6/index.html"><p>for further information (hood) click here</p></a>
+
<a href="http://www.avestin.com/gc3page.html"><p>for further information click here</p></a>
−
<a href="https://www.biostep.de/products/Bio_Imaging_software_1843/Acquisition_and_control_software_1844/argus_X1___for_cameras_and_scanners_1845/biostep_argusX1__basic_licence_1/index.html"><p>for further information (software) click here</p></a>
+
−
</html>
+
−
[[File:Darkhood.JPG|center|thumb]]
+
−
<html>
+
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Large Centrifuge</h3>
+
<h3>Eppi-shaker/Heatingblock</h3>
 
<ul>
 
<ul>
−
<li>Eppendorf Centrifuge 5810 R</li>
+
<li>Eppendorf Thermomixer compact</li>
−
<a href="http://www.eppendorf.com/int/index.php?pb=01edea0fe7a0e358&action=products&contentid=1&catalognode=10019"><p>for further information click here</p></a>
+
<a href="http://www.eppendorf.com/int/index.php?pb=93a83e2a7b7b1b05&action=products&contentid=1&catalognode=9840"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:BigCentrifuge.JPG|center|thumb]]
+
[[File:HeatingBlock.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Small Centrifuge</h3>
+
<h3>FACS</h3>
 
<ul>
 
<ul>
−
<li>Eppendorf Centrifuge 5424 R</li>
+
<li>BD LSRFortessa™ cell analyzer</li>
−
<a href="http://www.labdepotinc.com/Product_Details~id~450~pid~59833.aspx"><p>for further information click here</p></a>
+
<a href="http://www.bdbiosciences.com/us/instruments/research/cell-analyzers/bd-lsrfortessa/m/751752?cc=US"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:EppiCentrifuge.JPG|center|thumb]]
+
[[File:FACS.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Shaker</h3>
+
<h3>Fluorescence Scanner for Western Blots</h3>
 
<ul>
 
<ul>
−
<li>Kuhner Climo-Shaker ISF1-X</li>
+
<li>ODYSSEY® CLx Infrared Imaging System</li>
−
<a href="http://www.kuhner.com/de/produkte/schuettelmaschinen/inkubatoren/isf1-x.html"><p>for further information click here</p></a>
+
<a href="http://www.licor.com/bio/products/imaging_systems/odyssey/"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:ShakerIncubator.JPG|center|thumb]]
+
[[File:FluorescenceScanner.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>PCR Machine</h3>
+
<h3>Incubator</h3>
 
<ul>
 
<ul>
−
<li>Applied Biosystems Veriti 96Well Thermal Cycler</li>
+
<li>Thermo Scientific Heraeus Incubator</li>
−
<a href="https://www.thermofisher.com/ch/en/home/life-science/pcr/thermal-cyclers-realtime-instruments/thermal-cyclers/veriti-thermal-cycler.html"><p>for further information click here</p></a>
+
<a href="https://webshop.fishersci.com/insight2_uk/getProduct.do?productCode=11626250&resultSetPosition=0"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:PCRMachine.JPG|center|thumb]]
+
[[File:Incubator.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Gel Electrophoresis</h3>
+
<h3>Large Centrifuge</h3>
 
<ul>
 
<ul>
−
<li>Helixx Technologies MyRun®</li>
+
<li>Eppendorf Centrifuge 5810 R</li>
−
<a href="http://www.helixxtec.com/helixx/c/iMyRun/iMyRun.htm"><p>for further information click here</p></a>
+
<a href="http://www.eppendorf.com/int/index.php?pb=01edea0fe7a0e358&action=products&contentid=1&catalognode=10019"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:GelRunningThing.JPG|center|thumb]]
+
[[File:BigCentrifuge.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Eppi-shaker/Heatingblock</h3>
+
<h3>Nano Drop</h3>
 
<ul>
 
<ul>
−
<li>Eppendorf Thermomixer compact</li>
+
<li>Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c Software</li>
−
<a href="http://www.eppendorf.com/int/index.php?pb=93a83e2a7b7b1b05&action=products&contentid=1&catalognode=9840"><p>for further information click here</p></a>
+
<a href="http://www.nanodrop.com/Productnd2000overview.aspx"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:HeatingBlock.JPG|center|thumb]]
+
[[File:NanoDrop.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Vortex</h3>
+
<h3>PCR Machine</h3>
 
<ul>
 
<ul>
−
<li>Scientific Industries VortexGenie®2</li>
+
<li>Applied Biosystems Veriti 96Well Thermal Cycler</li>
−
<a href="http://www.scientificindustries.com/genie2.html"><p>for further information click here</p></a>
+
<a href="https://www.thermofisher.com/ch/en/home/life-science/pcr/thermal-cyclers-realtime-instruments/thermal-cyclers/veriti-thermal-cycler.html"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:Vortex.JPG|center|thumb]]
+
[[File:PCRMachine.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Balance</h3>
+
<h3>Plate Reader</h3>
 
<ul>
 
<ul>
−
<li>MettlerToledo XS4002S DeltaRange® Balance</li>
+
<li>Tecan Infinite M200 Pro™ combined with Tecan iControl TM software</li>
−
<a href="http://us.mt.com/us/en/home/products/Laboratory_Weighing_Solutions/Precision_Balances/XS_Precision_Balances/XS_4002_S_Dual_Range.html"><p>for further information click here</p></a>
+
<a href="http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1"><p>for further information (platereader) click here</p></a>
 +
<a href="http://www.tecan.ch/platform/apps/product/index.asp?MenuID=1817&ID=1924&Menu=1&Item=21.7.8"><p>for further information (software) click here</p></a>
 
</html>
 
</html>
−
[[File:Balance.JPG|center|thumb]]
+
[[File:TecanPlateReader.JPG|center|thumb]]
 +
<html>
 +
</html>
 +
[[File:ProgramTecanReader.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Incubator</h3>
+
<h3>Shaker</h3>
 
<ul>
 
<ul>
−
<li>Thermo Scientific Heraeus Incubator</li>
+
<li>Kuhner Climo-Shaker ISF1-X</li>
−
<a href="https://webshop.fishersci.com/insight2_uk/getProduct.do?productCode=11626250&resultSetPosition=0"><p>for further information click here</p></a>
+
<a href="http://www.kuhner.com/de/produkte/schuettelmaschinen/inkubatoren/isf1-x.html"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:Incubator.JPG|center|thumb]]
+
[[File:ShakerIncubator.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
Line 244: Line 222:
 
<div class="info">
 
<div class="info">
  
−
<h3>FACS</h3>
+
<h3>Small Centrifuge</h3>
 
<ul>
 
<ul>
−
<li>BD LSRFortessa™ cell analyzer</li>
+
<li>Eppendorf Centrifuge 5424 R</li>
−
<a href="http://www.bdbiosciences.com/us/instruments/research/cell-analyzers/bd-lsrfortessa/m/751752?cc=US"><p>for further information click here</p></a>
+
<a href="http://www.labdepotinc.com/Product_Details~id~450~pid~59833.aspx"><p>for further information click here</p></a>
 
</html>
 
</html>
−
[[File:FACS.JPG|center|thumb]]
+
[[File:EppiCentrifuge.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
−
 
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Fluorescence Scanner for Western Blots</h3>
+
<h3>UV- and Dark Hood</h3>
 
<ul>
 
<ul>
−
<li>ODYSSEY® CLx Infrared Imaging System</li>
+
<li>Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence</li>
−
<a href="http://www.licor.com/bio/products/imaging_systems/odyssey/"><p>for further information click here</p></a>
+
<a href="http://www.biostep.de/i18n_en_DE/products/Bio_Imaging_components_1776/Tripod__dark_hoods_and_accessories_1863/Dark_hoods_1865/Dark_Hood_DH_40_with_control_panel_and_display_6/index.html"><p>for further information (hood) click here</p></a>
 +
<a href="https://www.biostep.de/products/Bio_Imaging_software_1843/Acquisition_and_control_software_1844/argus_X1___for_cameras_and_scanners_1845/biostep_argusX1__basic_licence_1/index.html"><p>for further information (software) click here</p></a>
 
</html>
 
</html>
−
[[File:FluorescenceScanner.JPG|center|thumb]]
+
[[File:Darkhood.JPG|center|thumb]]
 
<html>
 
<html>
 
</ul>
 
</ul>
Line 273: Line 251:
 
<div class="info">
 
<div class="info">
  
−
<h3>Emulsifier</h3>
+
<h3>Vortex</h3>
 
<ul>
 
<ul>
−
<li>EmulsiFlex-C3 from AVESTIN, Inc. and AVESTIN Europe GmbH/li>
+
<li>Scientific Industries VortexGenie®2</li>
−
<a href="http://www.avestin.com/gc3page.html"><p>for further information click here</p></a>
+
<a href="http://www.scientificindustries.com/genie2.html"><p>for further information click here</p></a>
 +
</html>
 +
[[File:Vortex.JPG|center|thumb]]
 +
<html>
 
</ul>
 
</ul>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 
</div>
 
</div>
 +
  
 
</div>
 
</div>

Revision as of 16:02, 8 September 2015

"What I cannot create I do not understand."
- Richard Feynmann

Materials

Bacterial strains and Mammalian Cell Lines

TOP10

The bacterial strain we used as a base for all our transformations was TOP10.

A list of all strains we generated in the course of our project can be found here (link).

Jurkat Cells

Immortalized human T lymphocyte cell line. We used this cell line as a model line for cancer cells with elevated lactate production and TRAIL susceptibility (papers).

HL60

Cell line derived from acute myeloid leukemia. We used this cell line as a model line for cancer cells with elevated lactate production and TRAIL susceptibility (papers).

SK-BR-3

HER2-positive breast cancer cell line. We used this cell line to validate the chimeric receptors of the Stockholm iGEM team.

3T3

Murine fibroblast cell line. We used this cell line as a model line for healthy cells which are restistant to TRAIL and produce normal levels of lactate.

Machines

Absorbance Measurments

Balance

Gel Electrophoresis

Emulsifier

Eppi-shaker/Heatingblock

FACS

Fluorescence Scanner for Western Blots

Incubator

Large Centrifuge

Nano Drop

  • Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c Software

    • for further information click here

    Error creating thumbnail: Invalid thumbnail parameters

PCR Machine

Plate Reader

Shaker

Small Centrifuge

UV- and Dark Hood

Vortex

Buffers and media

TFP1

  • 100 mM RbCl
  • 50 mM MnCl2
  • 30 mM potassium acetate
  • 10 mM CaCl2
  • 15%glycerol

TFP2

  • 100 mM MOPS
  • 50 mM RbCl
  • 75 mM CaCl2
  • 15%glycerol

SOC medium

  • 20 g/L Tryptophane
  • 5 g/L Yeast extract
  • 0.5 g/L NaCl
  • 250 mM KCl
  • 1M MgCl2
  • 50% (w/v) sterile glucose
  • Filter the solution through a 0.2 µm filter.

3T3 medium

  • 500 mL D-MEM 1x
  • 50 mL FBS 100%
  • 5 mL GlutaMax 100x
  • 5 mL Penicillin (10k units/mL) /Streptomyocin (10k µg/mL )

Jurkat medium

  • 2 mL MEM 1x
  • 20 mL FBS 100%
  • 2 mL GlutaMax 100x
  • 0.4 mL Penicillin (10k units/mL) /Streptomyocin (10k µg/mL )
  • 200 mL RPMI

Annexin V binding buffer

  • 10 mM HEPES
  • 140 mM NaCl
  • 2.5 mM
  • 250 mM CaCl2
  • pH 7.4
  • Filter the solution through a 0.2 µm filter.

Chemicals

LB-Broth

Agarose

Agar

Glycerol

2-log DNA ladder

Taq DNA Ligase

Cutsmart Buffer

EcoRI

DpnI

Q5 Master Mix

dNTP Mix

Taq DNA Polymerase

AnnexinV-Alexa

Green DNA/RNA Dye

Streptavidin Beads

Red Gel Stain

Anti-Human-AnnexinV

Gel Loading Dye

T4 DNA Ligase

PstI

DNA Ligase Buffer

Ethanol

DMEM

DMEM with Glutamine

RPMI

Accutase

Kits

Plasmid Purification

  • Product: ZR Plasmid Miniprep ™-Classic
  • Company: Zymo Research
  • Lot: ZRC182892
  • Catalog No: D4016

Changes to the protocol included an extra step to dry the column before elution and the use of water for elution instead of elution buffer. For elution of large plasmids we used prewarmed water.

Gel Extraction

  • Product: ZymocleanTM Gel DNA Recovery Kit
  • Company: Zymo Research
  • Lot: ZRC183055
  • Catalog No: D4002

Changes to the protocol included an extra step to dry the column before elution and the use of water for elution instead of elution buffer. For elution of large plasmids we used prewarmed water.

Lactate Detection Kit

  • Product: L-Lactic Acid
  • Company: Megazyme
  • Lot:
  • Catalog No:

Changes to the protocol included an extra step to dry the column before elution and the use of water for elution instead of elution buffer. For elution of large plasmids we used prewarmed water.

The detection of lactate using this kit relies on two enzymatic reactions. In the first, L-lactate and NAD&sup+; were converted into pyruvate and NADH+H&sup+; by L-lactate dehydrogenase. In the coupled reaction, pyruvate is used with D-glutamate by the D-glutamate-pyruvate transaminase to have the equilibrium of the first reaction in favour of the pyruvate. The concentration of NADH is finally measured as the optical density at 340 nm (OD340).