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

 
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<h1>Materials</h1>
 
<h1>Materials</h1>
  
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<p></p>
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<p></p>
 +
 +
 +
<h2>Bacterial strains and Mammalian Cell Lines</h2>
 +
 +
<h3><i>Escherichia coli</i> TOP10</h3>
 +
<p>The bacterial strain we used as a base for all our transformations was TOP10 (Genotype: F- mcrA &Delta;(mrr-hsdRMS-mcrBC) &phi;80lacZ&Delta;M15 &Delta;lacX74 nupG recA1 araD139 &Delta;(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 &lambda;-). These cells were provided by the Panke Group at ETH Zurich.</p>
 +
 +
 +
<h3>Jurkat Cells</h3>
 +
 +
<p>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). A big thank you to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.</p>
 +
 +
<h3>HL60</h3>
 +
 +
<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). Many thanks to the Neri Group at ETH Zurich who kindly offered us an aliquot of their cells.</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. We are very grateful to Michal Stanczak from the University Hospital of Basel for offering us a sample of this cell line.</p>
 +
 +
<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. Many thanks to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.</p>
 +
 +
<h3>3T3 P65 -/- </h3>
 +
<p>Murine fluorescent fibroblast cell line.
 +
<ul>
 +
<li> H2B-GFP: nuclear staining</li>
 +
<li> P65- dsRed: cytoskeleton</li>
 +
</ul>
 +
We used this cell line to study the co-culture of the bacteria and the chip. Many thanks to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.</p>
 +
</div>
 +
 +
 +
 +
 +
 +
 +
<div class="expContainer">
  
 
<h2>Buffers and media </h2>
 
<h2>Buffers and media </h2>
 +
 
<div class="info">
 
<div class="info">
−
<h3>TFP1</h3>
+
<h3>Annexin V binding buffer</h3>
 
<ul>
 
<ul>
−
<li>100 mM RbCl</li>
+
<li>10 mM HEPES</li>
−
<li>50 mM MnCl2</li>
+
<li>140 mM NaCl</li>
−
<li>30 mM potassium acetate</li>
+
<li>2.5 mM</li>
−
<li>10 mM CaCl<sub>2</sub></li>
+
<li>250 mM CaCl<sub>2</sub></li>
−
<li>15&#37;glycerol</li>
+
<li>pH 7.4</li>
 +
<li>Filter the solution through a 0.2 &micro;m filter. </li>
 
</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>Jurkat medium</h3>
 +
<ul>
 +
<li>2 mL MEM 1x</li>
 +
<li>20 mL FBS 100%</li>
 +
<li>2 mL GlutaMax 100x</li>
 +
<li>0.4 mL Penicillin (10k units/mL) /Streptomyocin (10k &micro;g/mL )</li>
 +
<li>200 mL RPMI</li>
 +
</ul>
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
−
<h3>TFP2</h3>
+
<div class="info">
 +
 
 +
<h3>Terrific Broth</h3>
 
<ul>
 
<ul>
−
<li>100 mM MOPS</li>
+
<li>2.32 g/L KH<sub>2</sub>PO<sub>4</sub></li>
−
<li>50 mM RbCl</li>
+
<li>12.54 g/L K<sub>2</sub>HPO<sub>4</sub></li>
−
<li>75 mM CaCl<sub>2</sub></li>
+
<li>1.2% Tryptone</li>
−
<li>15&#37;glycerol</li>
+
<li>2.4% Yeast Extract</li>
 +
<li>0.4% Gylcerol</li>
 +
<li>Filter the solution through a 0.2 &micro;m filter.</li>
 
</ul>
 
</ul>
  
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<li>0.5 g/L NaCl </li>
 
<li>0.5 g/L NaCl </li>
 
<li>250 mM KCl</li>
 
<li>250 mM KCl</li>
−
<li>1M MgCl<sub>2</sub></li>
+
<li>1 M MgCl<sub>2</sub></li>
 
<li>50&#37; (w/v) sterile glucose</li>
 
<li>50&#37; (w/v) sterile glucose</li>
 
<li>Filter the solution through a 0.2 &micro;m filter. </li>
 
<li>Filter the solution through a 0.2 &micro;m filter. </li>
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<div class="info">
 
<div class="info">
−
 
+
<h3>TFB1</h3>
−
<h3>3T3 medium</h3>
+
 
<ul>
 
<ul>
−
<li>500 mL D-MEM 1x</li>
+
<li>100 mM RbCl</li>
−
<li>50 mL FBS 100%</li>
+
<li>50 mM MnCl2</li>
−
<li>5 mL GlutaMax 100x</li>
+
<li>30 mM potassium acetate</li>
−
<li>5 mL Penicillin (10k units/mL) /Streptomyocin (10k &micro;g/mL )</li>
+
<li>10 mM CaCl<sub>2</sub></li>
 +
<li>15&#37; glycerol</li>
 
</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>
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<div class="info">
 
<div class="info">
  
−
<h3>Jurkat medium</h3>
+
 
 +
<h3>TFB2</h3>
 
<ul>
 
<ul>
−
<li>2 mL MEM 1x</li>
+
<li>100 mM MOPS</li>
−
<li>20 mL FBS 100%</li>
+
<li>50 mM RbCl</li>
−
<li>2 mL GlutaMax 100x</li>
+
<li>75 mM CaCl<sub>2</sub></li>
−
<li>0.4 mL Penicillin (10k units/mL) /Streptomyocin (10k &micro;g/mL )</li>
+
<li>15&#37; glycerol</li>
−
<li>200 mL RPMI</li>
+
 
</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>Annexin V binding buffer</h3>
+
<h3>3T3 medium</h3>
 
<ul>
 
<ul>
−
<li>10 mM HEPES</li>
+
<li>500 mL D-MEM 1x</li>
−
<li>140 mM NaCl</li>
+
<li>50 mL FBS 100%</li>
−
<li>2.5 mM</li>
+
<li>5 mL GlutaMax 100x</li>
−
<li>250 mM CaCl<sub>2</sub></li>
+
<li>5 mL Penicillin (10k units/mL) /Streptomyocin (10k &micro;g/mL )</li>
−
<li>pH 7.4</li>
+
−
<li>Filter the solution through a 0.2 &micro;m filter. </li>
+
 
</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>
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<div class="expContainer">
 
<div class="expContainer">
  
−
<h2>Chemicals and Kits</h2>
+
<h2>Chemicals</h2>
  
 
<div class="info">
 
<div class="info">
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<h3>LB-Broth</h3>
 
<h3>LB-Broth</h3>
 
<ul>
 
<ul>
−
     <li>Product name: Difco™ LB Broth, Miller (Lurica-Bertani)</li>
+
     <li>Product name: BD Difco™ LB Broth, Miller (Luria-Bertani)</li>
 
     <li>Company: BD</li>     
 
     <li>Company: BD</li>     
−
     <li>Lot: 4339957</li>
+
     <li>REF: 244620</li>
−
     <li>CAS: </li>
+
     <a href="http://catalog.bd.com/nexus-ecat/getProductDetail?productId=244620&parentCategory=1211&parentCategoryName=Microbiology%20Testing&categoryId=1269&categoryName=Dehydrated%20Culture%20Media&searchUrl="><p>for further information click here</p></a>
−
    <li><a href=""><p>for further information click here</p></a></li>
+
 
</ul>
 
</ul>
  
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     <li>Product name: Agarose</li>
 
     <li>Product name: Agarose</li>
 
     <li>Company: Sigma-Aldrich</li>
 
     <li>Company: Sigma-Aldrich</li>
−
     <li>Lot: #SLBK3392V</li>
+
     <li>REF: A9539-500G</li>
−
     <li>CAS: 9012-36-6</li>
+
    <a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9539?lang=de&region=CH"><p>for further information click here</p></a>
−
     <li><a href=""><p>for further information click here</p></a></li>
+
</ul>
 +
 
 +
<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 class="info">
 +
 
 +
<h3>Agar</h3>
 +
<ul>
 +
    <li>Product name: Agar für die Bakteriologie BC</li>
 +
     <li>Company: Sigma-Aldrich</li>
 +
     <li>REF: A0949</li>
 +
    <a href="https://www.applichem.com/shop/produktdetail/as/agar-fuer-die-bakteriologie/"><p>for further information click here</p></a>
 
</ul>
 
</ul>
  
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<h3>Glycerol</h3>
 
<h3>Glycerol</h3>
 
<ul>
 
<ul>
−
     <li>Product: Glycerol anhydrous BioChemica</li>
+
     <li>Product: Glycerol anhydrous BioChemica BC</li>
 
     <li>Company: AppliChem</li>
 
     <li>Company: AppliChem</li>
−
     <li>Lot: </li>
+
     <li>REF: A1123</li>
−
     <li>CAS: </li>
+
     <a href="https://www.applichem.com/en/shop/product-detail/as/glycerin-wasserfrei-ibiochemicai/"><p>for further information click here</p></a>
−
    <li><a href=""><p>for further information click here</p></a></li>
+
 
</ul>
 
</ul>
  
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     <li>Company: New England BioLabs</li>
 
     <li>Company: New England BioLabs</li>
 
     <li>REF: N3200S</li>
 
     <li>REF: N3200S</li>
−
     <li><a href="https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb"><p>for further information click here</p></a></li>
+
     <a href="https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb"><p>for further information click here</p></a>
 
</ul>
 
</ul>
  
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<div class="info">
 
<div class="info">
  
−
<h3></h3>
+
<h3>Taq DNA Ligase</h3>
 
<ul>
 
<ul>
−
     <li>Product: </li>
+
     <li>Product: Taq DNA Ligase </li>
−
     <li>Company: </li>
+
     <li>Company: New England BioLabs</li>
−
     <li>CAS: </li>
+
     <li>REF: M0208S</li>
−
     <li><a href=""><p>for further information click here</p></a></li>
+
     <a href="https://www.neb.com/products/m0208-taq-dna-ligase"><p>for further information click here</p></a>
 
</ul>
 
</ul>
  
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<div class="info">
 
<div class="info">
  
−
<h3></h3>
+
<h3>Cutsmart Buffer</h3>
 
<ul>
 
<ul>
−
     <li>Product: </li>
+
     <li>Product: CutSmart® Buffer </li>
−
     <li>Company: </li>
+
     <li>Company: New England BioLabs</li>
−
     <li>CAS: </li>
+
     <li>REF: B7204S</li>
−
     <li><a href=""><p>for further information click here</p></a></li>
+
     <a href="https://www.neb.com/products/b7204-cutsmart-buffer"><p>for further information click here</p></a>
 
</ul>
 
</ul>
  
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<div class="info">
 
<div class="info">
  
−
<h3></h3>
+
<h3>EcoRI</h3>
 
<ul>
 
<ul>
−
     <li>Product: </li>
+
     <li>Product: EcoRI-HF® </li>
−
     <li>Company: </li>
+
     <li>Company: New England BioLabs</li>
−
     <li>CAS: </li>
+
     <li>REF: R3101S</li>
−
     <li><a href=""><p>for further information click here</p></a></li>
+
     <a href="https://www.neb.com/products/r3101-ecori-hf"><p>for further information click here</p></a>
 
</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></h3>
+
<h3>BsaI-HF</h3>
 
<ul>
 
<ul>
−
     <li>Product: </li>
+
     <li>Product: BsaI-HF® </li>
−
     <li>Company: </li>
+
     <li>Company: New England BioLabs</li>
−
     <li>Lot: </li>
+
     <li>REF: R3535S</li>
−
     <li>CAS: </li>
+
     <a href="https://www.neb.com/products/r3535-bsai-hf"><p>for further information click here</p></a>
 
</ul>
 
</ul>
  
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 +
<div class="info">
  
 +
<h3>DpnI</h3>
 +
<ul>
 +
    <li>Product: DpnI</li>
 +
    <li>Company: New England BioLabs</li>
 +
    <li>REF: R0176S</li>
 +
    <a href="https://www.neb.com/products/r0176-dpni"><p>for further information click here</p></a>
 +
</ul>
  
 +
<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 class="info">
 
<div class="info">
  
−
<h3>Plasmid Purification</h3>
+
<h3>Q5 Master Mix</h3>
−
 
+
 
<ul>
 
<ul>
−
     <li>Product: ZR Plasmid Miniprep ™-Classic</li>
+
     <li>Product: Q5® High-Fidelity 2X Master Mix </li>
−
     <li>Company: Zymo Research</li>
+
     <li>Company: New England BioLabs</li>
−
     <li>Lot: ZRC182892</li>
+
     <li>REF: M0492S</li>
−
     <li>Catalog No: D4016</li>
+
     <a href="https://www.neb.com/products/m0492-q5-high-fidelity-2x-master-mix"><p>for further information click here</p></a>
 
</ul>
 
</ul>
−
 
−
<p>
 
−
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.
 
−
</p>
 
  
 
<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>
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<div class="info">
 
<div class="info">
  
−
<h3>Gel Extraction</h3>
+
<h3>dNTP Mix</h3>
 +
<ul>
 +
    <li>Product: Deoxynucleotide (dNTP) Solution Mix </li>
 +
    <li>Company: New England BioLabs</li>
 +
    <li>REF: N1201A</li>
 +
    <a href="https://www.neb.com/~/media/Catalog/All-Products/690D917CFEBC46C689678BC243668395/MSDS/sdsN1201-cae.pdf"><p>for further information click here</p></a>
 +
</ul>
  
 +
<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 class="info">
 +
 +
<h3>Taq DNA Polymerase</h3>
 
<ul>
 
<ul>
−
     <li>Product: ZymocleanTM Gel DNA Recovery Kit</li>
+
     <li>Product: Taq DNA Polymerase with ThermoPol® Buffer </li>
−
     <li>Company: Zymo Research</li>
+
     <li>Company: New England BioLabs</li>
−
     <li>Lot: ZRC183055</li>
+
     <li>REF: M0276L</li>
−
     <li>Catalog No: D4002</li>
+
     <a href="https://www.neb.com/products/m0267-taq-dna-polymerase-with-thermopol-buffer"><p>for further information click here</p></a>
 
</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>
 +
</div>
  
−
<p>
+
 
−
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.
+
<div class="info">
−
</p>
+
 
 +
<h3>AnnexinV-Alexa</h3>
 +
<ul>
 +
    <li>Product: Annexin V, Alexa Fluor® 488 conjugate </li>
 +
    <li>Company: ThermoFischer Scientific</li>
 +
    <li>REF: A13201</li>
 +
    <a href="https://www.thermofisher.com/order/catalog/product/A13201"><p>for further information click here</p></a>
 +
</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>
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<div class="info">
 
<div class="info">
  
−
<h3>Lactate Detection Kit</h3>
+
<h3>Green DNA/RNA Dye</h3>
 
<ul>
 
<ul>
−
     <li>Product: L-Lactic Acid</li>
+
     <li>Product: peqGREEN</li>
−
     <li>Company: Megazyme</li>
+
     <li>Company: peqlab</li>
−
     <li>Lot: </li>
+
     <li>REF: 37-5010</li>
−
     <li>Catalog No: </li>
+
     <a href="http://shop.axonlab.ch/D/include/ShowPDFDocument.asp?SessionId=700C97586C134BCEA1C5C30FBD553A03&adilng=D&ArtNr=37-5010&Nummer=3"><p>for further information click here</p></a>
 
</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>
 +
</div>
  
−
<p>
+
<div class="info">
−
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.
+
 
−
</p>
+
<h3>Protein G coated Beads</h3>
−
+
<ul>
−
<p>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).</p>
+
    <li>Product: Pierce™ Protein G Magnetic Beads</li>
 +
    <li>Company: Thermo Fischer Scientific</li>
 +
    <li>REF: 88847</li>
 +
    <a href="https://www.thermofisher.com/order/catalog/product/88847"><p>for further information click here</p></a>
 +
</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">
  
 +
<h3>Streptavidin Beads</h3>
 +
<ul>
 +
    <li>Product: Roti®-MagBeads Streptavidin</li>
 +
    <li>Company: Carl Roth</li>
 +
    <li>REF: HP57.2</li>
 +
    <a href="https://www.carlroth.com/de/de/Chemikalien/A-Z-Chemikalien/R/Roti%3Csup%3E%C2%AE%3C-sup%3E-MagBeads/Roti%3Csup%3E%C2%AE%3C-sup%3E-MagBeads-Streptavidin/p/0000000a0002a23a00010023_de"><p>for further information click here</p></a>
 +
</ul>
 +
 +
<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="expContainer">
+
<div class="info">
  
 +
<h3>Red Gel Stain</h3>
 +
<ul>
 +
    <li>Product: GelRed Nucleic Acid Gel Stain, 10,000X in water </li>
 +
    <li>Company: Biotium</li>
 +
    <li>REF: 41003</li>
 +
    <a href="https://biotium.com/product/gelredtm-nucleic-acid-gel-stain-10000x-in-water/"><p>for further information click here</p></a>
 +
</ul>
  
−
<h2>Bacterial strains and Mammalian Cell Lines</h2>
+
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
−
<h3>TOP10</h3>
 
−
<p>The bacterial strain we used as a base for all our transformations was TOP10.</p>
 
  
−
<p>A list of all strains we generated in the course of our project can be found here (link).</p>
 
  
−
<h3>Jurkat Cells</h3>
+
<div class="info">
  
−
<p>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).</p>
+
<h3>Anti-Human-AnnexinV</h3>
 +
<ul>
 +
    <li>Product: anti-human Annexin V/Biotin </li>
 +
    <li>Company: Affymetrix eBioscience</li>
 +
    <li>REF: BMS147</li>
 +
    <a href="http://www.ebioscience.com/human-annexin-v-antibody-biotin-vaa-33.htm"><p>for further information click here</p></a>
 +
</ul>
  
−
<h3>HL60</h3>
+
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
−
<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>3T3</h3>
+
<div class="info">
  
−
<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>Gel Loading Dye</h3>
 +
<ul>
 +
    <li>Product: Gel Loading Dye, Purple (6X), no SDS </li>
 +
    <li>Company: New England BioLabs</li>
 +
    <li>REF: B7025S</li>
 +
    <a href="https://www.neb.com/products/b7025-gel-loading-dye-purple-6x-no-sds"><p>for further information click here</p></a>
 +
</ul>
  
−
<h3>SK-BR-3</h3>
+
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
−
<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 class="info">
 +
 +
<h3>T4 DNA Ligase</h3>
 +
<ul>
 +
    <li>Product: T4 DNA Ligase</li>
 +
    <li>Company: New England BioLabs</li>
 +
    <li>REF: M0202S</li>
 +
    <a href="https://www.neb.com/products/m0202-t4-dna-ligase"><p>for further information click here</p></a>
 +
</ul>
 +
 +
<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="expContainer">
+
<div class="info">
  
−
<h2>Machines</h2>
+
<h3>PstI</h3>
 +
<ul>
 +
    <li>Product: PstI</li>
 +
    <li>Company: New England BioLabs</li>
 +
    <li>REF: M0140S</li>
 +
    <a href="https://www.neb.com/products/r0140-psti"><p>for further information click here</p></a>
 +
</ul>
 +
 
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
  
Line 324: Line 486:
 
<div class="info">
 
<div class="info">
  
−
<h3>Nano Drop</h3>
+
<h3>DNA Ligase Buffer</h3>
 
<ul>
 
<ul>
−
<li>Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c Software</li>
+
    <li>Product: Taq DNA Ligase Reaction Buffer </li>
−
<a href="http://www.nanodrop.com/Productnd2000overview.aspx"><p>for further information click here</p></a>
+
    <li>Company: New England BioLabs</li>
−
</html>
+
    <li>REF: B0208S</li>
−
[[File:NanoDrop.JPG|center|thumb]]
+
    <a href="https://www.neb.com/products/b0208-taq-dna-ligase-reaction-buffer"><p>for further information click here</p></a>
−
<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">
 +
 +
<h3>Ethanol</h3>
 +
<ul>
 +
    <li>Product: Ethanol </li>
 +
    <li>Company: Sigma-Aldrich</li>
 +
    <li>REF: 02860-2.5L</li>
 +
    <a href="http://www.sigmaaldrich.com/catalog/product/fluka/02860?lang=de&region=CH"><p>for further information click here</p></a>
 +
</ul>
 +
 +
<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 class="info">
 
<div class="info">
  
−
<h3>Plate Reader</h3>
+
<h3>DMEM</h3>
 
<ul>
 
<ul>
−
<li>Tecan Infinite M200 Pro™ combined with Tecan iControl TM software</li>
+
    <li>Product: DMEM, high glucose, no glutamine, no phenol red</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>
+
    <li>Company: Gibco ThermoFisher Scientific</li>
−
<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>
+
    <li>REF: 31053-028</li>
−
</html>
+
    <a href="https://www.thermofisher.com/order/catalog/product/31053028"><p>for further information click here</p></a>
−
[[File:TecanPlateReader.JPG|center|thumb]]
+
−
<html>
+
−
</html>
+
−
[[File:ProgramTecanReader.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>
Line 359: Line 529:
 
<div class="info">
 
<div class="info">
  
−
<h3>Absorbance Measurments</h3>
+
<h3>DMEM with Glutamine</h3>
 
<ul>
 
<ul>
−
<li>Novaspec Plus™ Visible Spectrophotometer</li>
+
    <li>Product: DMEM cell culture media</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>
+
    <li>Company: Gibco ThermoFisher Scientific</li>
−
</html>
+
    <li>REF: 41965-039</li>
−
[[File:ODPhotometer.JPG|center|thumb]]
+
    <a href="http://wiki.toxbank.net/wiki/DMEM_cell_culture_media_%2841965-039%29"><p>for further information click here</p></a>
−
<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 374: Line 544:
 
<div class="info">
 
<div class="info">
  
−
<h3>UV- and Dark Hood</h3>
+
<h3>RPMI</h3>
 
<ul>
 
<ul>
−
<li>Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence</li>
+
    <li>Product: RPMI 1640 Medium, no phenol red</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>
+
    <li>Company: Gibco ThermoFisher Scientific</li>
−
<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>
+
    <li>REF: 11835-063</li>
−
</html>
+
    <a href="https://www.thermofisher.com/order/catalog/product/11835063"><p>for further information click here</p></a>
−
[[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>
Line 390: Line 559:
 
<div class="info">
 
<div class="info">
  
−
<h3>Large Centrifuge</h3>
+
<h3>Accutase</h3>
 
<ul>
 
<ul>
−
<li>Eppendorf Centrifuge 5810 R</li>
+
    <li>Product: StemPro® Accutase® Cell Dissociation Reagent</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>
+
    <li>Company: Gibco ThermoFisher Scientific</li>
−
</html>
+
    <li>REF: A11105-01</li>
−
[[File:BigCentrifuge.JPG|center|thumb]]
+
    <a href="https://www.thermofisher.com/order/catalog/product/A1110501"><p>for further information click here</p></a>
−
<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">
 +
 +
<h3>SuperKillerTRAIL™</h3>
 +
<ul>
 +
    <li>Product: SuperKillerTRAIL™ </li>
 +
    <li>Company: Enzo</li>
 +
    <li>REF: N3200SALX-201-115-C010</li>
 +
    <a href="http://www.enzolifesciences.com/ALX-201-115/superkillertrail-soluble-human-recombinant/"><p>for further information click here</p></a>
 +
</ul>
 +
 +
<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 class="info">
 
<div class="info">
  
−
<h3>Small Centrifuge</h3>
+
<h3>TRAIL</h3>
 
<ul>
 
<ul>
−
<li>Eppendorf Centrifuge 5424 R</li>
+
    <li>Product: TRAIL / APO2 Ligand Human Recombinant </li>
−
<a href="http://www.labdepotinc.com/Product_Details~id~450~pid~59833.aspx"><p>for further information click here</p></a>
+
    <li>Company: ProspecBio</li>
−
</html>
+
    <li>REF: CYT-443</li>
−
[[File:EppiCentrifuge.JPG|center|thumb]]
+
    <a href="http://www.prospecbio.com/APO2L_Human_3_9/"><p>for further information click here</p></a>
−
<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 class="expContainer">
 +
 +
<h2>Kits</h2>
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Shaker</h3>
+
<h3>Plasmid Purification</h3>
 +
 
 
<ul>
 
<ul>
−
<li>Kuhner Climo-Shaker ISF1-X</li>
+
    <li>Product: ZR Plasmid Miniprep ™-Classic</li>
−
<a href="http://www.kuhner.com/de/produkte/schuettelmaschinen/inkubatoren/isf1-x.html"><p>for further information click here</p></a>
+
    <li>Company: Zymo Research</li>
−
</html>
+
    <li>Lot: ZRC182892</li>
−
[[File:ShakerIncubator.JPG|center|thumb]]
+
    <li>Catalog No: D4016</li>
−
<html>
+
 
</ul>
 
</ul>
 +
 +
<p>
 +
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.
 +
</p>
 +
 
<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">
 +
 +
<h3>Gel Extraction</h3>
 +
 +
 +
<ul>
 +
    <li>Product: ZymocleanTM Gel DNA Recovery Kit</li>
 +
    <li>Company: Zymo Research</li>
 +
    <li>Lot: ZRC183055</li>
 +
    <li>Catalog No: D4002</li>
 +
</ul>
 +
 +
 +
<p>
 +
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.
 +
</p>
 +
 +
<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 class="info">
 
<div class="info">
  
−
<h3>PCR Machine</h3>
+
<h3>Lactate Detection Kit</h3>
 
<ul>
 
<ul>
−
<li>Applied Biosystems Veriti 96Well Thermal Cycler</li>
+
    <li>Product: L-Lactic Acid</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>
+
    <li>Company: Megazyme</li>
−
</html>
+
    <li>Lot: </li>
−
[[File:PCRMachine.JPG|center|thumb]]
+
    <li>Catalog No: </li>
−
<html>
+
 
</ul>
 
</ul>
 +
 +
 +
<p>
 +
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.
 +
</p>
 +
 +
<p>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).</p>
 +
 
<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 class="expContainer">
 +
 +
<h2>Machines</h2>
 +
 +
<div class="info">
 +
 +
<h3>Absorbance Measurments</h3>
 +
<ul>
 +
<li>Novaspec Plus™ Visible Spectrophotometer</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>
 +
</ul>
 +
<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 class="info">
 +
 +
<h3>Balance</h3>
 +
<ul>
 +
<li>MettlerToledo XS4002S DeltaRange® Balance</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>
 +
</ul>
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
  
Line 454: Line 698:
 
<li>Helixx Technologies MyRun®</li>
 
<li>Helixx Technologies MyRun®</li>
 
<a href="http://www.helixxtec.com/helixx/c/iMyRun/iMyRun.htm"><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>
 
−
[[File:GelRunningThing.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">
 +
 +
<h3>Emulsifier</h3>
 +
<ul>
 +
<li>EmulsiFlex-C3 from AVESTIN, Inc. and AVESTIN Europe GmbH/li>
 +
<a href="http://www.avestin.com/gc3page.html"><p>for further information click here</p></a>
 +
</ul>
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
 +
</div>
  
  
Line 469: Line 720:
 
<li>Eppendorf Thermomixer compact</li>
 
<li>Eppendorf Thermomixer compact</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.eppendorf.com/int/index.php?pb=93a83e2a7b7b1b05&action=products&contentid=1&catalognode=9840"><p>for further information click here</p></a>
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</div>
 
</div>
 +
 +
 +
<div class="info">
 +
 +
<h3>FACS</h3>
 +
<ul>
 +
<li>BD LSRFortessa™ cell analyzer</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>
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</ul>
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<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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<div class="info">
  
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<h3>Vortex</h3>
+
<h3>Incubator</h3>
 
<ul>
 
<ul>
−
<li>Scientific Industries VortexGenie®2</li>
+
<li>Thermo Scientific Heraeus Incubator</li>
−
<a href="http://www.scientificindustries.com/genie2.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>
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</ul>
 
</ul>
 
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</div>
 
</div>
  
 +
 +
<div class="info">
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 +
<h3>Large Centrifuge</h3>
 +
<ul>
 +
<li>Eppendorf Centrifuge 5810 R</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>
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</ul>
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<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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<div class="info">
  
−
<h3>Balance</h3>
+
<h3>Nano Drop</h3>
 
<ul>
 
<ul>
−
<li>MettlerToledo XS4002S DeltaRange® Balance</li>
+
<li>Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c 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.nanodrop.com/Productnd2000overview.aspx"><p>for further information click here</p></a>
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<div class="info">
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<h3>PCR Machine</h3>
 +
<ul>
 +
<li>Applied Biosystems Veriti 96Well Thermal Cycler</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>
 +
</ul>
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<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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</div>
  
  
 
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<div class="info">
  
−
<h3>Incubator</h3>
+
<h3>Plate Reader</h3>
 
<ul>
 
<ul>
−
<li>Thermo Scientific Heraeus Incubator</li>
+
<li>Tecan Infinite M200 Pro™ combined with Tecan iControl TM software</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.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>
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</html>
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<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>
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[[File:Incubator.JPG|center|thumb]]
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</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>
  
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<div class="info">
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<h3>Shaker</h3>
 +
<ul>
 +
<li>Kuhner Climo-Shaker ISF1-X</li>
 +
<a href="http://www.kuhner.com/de/produkte/schuettelmaschinen/inkubatoren/isf1-x.html"><p>for further information click here</p></a>
 +
</ul>
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<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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<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>
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</ul>
 
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<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">
 +
 +
<h3>UV- and Dark Hood</h3>
 +
<ul>
 +
<li>Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence</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="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>
 +
</ul>
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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</div>
  
  
 
<div class="info">
 
<div class="info">
  
−
<h3>Fluorescence Scanner for Western Blots</h3>
+
<h3>Vortex</h3>
 
<ul>
 
<ul>
−
<li>ODYSSEY® CLx Infrared Imaging System</li>
+
<li>Scientific Industries VortexGenie®2</li>
 +
<a href="http://www.scientificindustries.com/genie2.html"><p>for further information click here</p></a>
 +
</ul>
 +
<a class="expander" href="#" onclick="expand(this);return false;"><img src="https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png"></a>
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 +
<div class="info">
 +
 
 +
<h3>LI-COR Odyssey®</h3>
 +
<ul>
 +
<li>LI-COR Odyssey® CLx infrared Western Blot Scanner</li>
 
<a href="http://www.licor.com/bio/products/imaging_systems/odyssey/"><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>
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{{:Template:ETH_Zurich/footer}}

Latest revision as of 23:53, 18 September 2015

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

Materials

Bacterial strains and Mammalian Cell Lines

Escherichia coli TOP10

The bacterial strain we used as a base for all our transformations was TOP10 (Genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-). These cells were provided by the Panke Group at ETH Zurich.

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). A big thank you to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.

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). Many thanks to the Neri Group at ETH Zurich who kindly offered us an aliquot of their cells.

SK-BR-3

HER2-positive breast cancer cell line. We used this cell line to validate the chimeric receptors of the Stockholm iGEM team. We are very grateful to Michal Stanczak from the University Hospital of Basel for offering us a sample of this cell line.

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. Many thanks to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.

3T3 P65 -/-

Murine fluorescent fibroblast cell line.

  • H2B-GFP: nuclear staining
  • P65- dsRed: cytoskeleton
We used this cell line to study the co-culture of the bacteria and the chip. Many thanks to the Tay Group at ETH Zurich who kindly offered us an aliquot of their cells.

Buffers and media

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.

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

Terrific Broth

  • 2.32 g/L KH2PO4
  • 12.54 g/L K2HPO4
  • 1.2% Tryptone
  • 2.4% Yeast Extract
  • 0.4% Gylcerol
  • Filter the solution through a 0.2 µm filter.

SOC medium

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

TFB1

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

TFB2

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

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 )

Chemicals

LB-Broth

Agarose

Agar

Glycerol

2-log DNA ladder

Taq DNA Ligase

Cutsmart Buffer

EcoRI

BsaI-HF

DpnI

Q5 Master Mix

dNTP Mix

Taq DNA Polymerase

AnnexinV-Alexa

Green DNA/RNA Dye

Protein G coated Beads

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

SuperKillerTRAIL™

TRAIL

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).

Machines

Absorbance Measurments

Balance

Gel Electrophoresis

Emulsifier

Eppi-shaker/Heatingblock

FACS

Incubator

Large Centrifuge

Nano Drop

PCR Machine

Plate Reader

Shaker

Small Centrifuge

UV- and Dark Hood

Vortex

LI-COR Odyssey®

We would like to thank our sponsors