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

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     <li>Product: peqGREEN</li>
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     <li>Company: peqlab</li>
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    <li>REF: 37-5010</li>
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    <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></li>
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<h3>Streptavidin Beads</h3>
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    <li>Product: Roti®-MagBeads Streptavidin</li>
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    <li>Company: Carl Roth</li>
 
     <li>REF: </li>
 
     <li>REF: </li>
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     <li><a href=""><p>for further information click here</p></a></li>
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     <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></li>
 
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<h3>Red Gel Stain</h3>
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<ul>
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    <li>Product: GelRed Nucleic Acid Gel Stain, 10,000X in water </li>
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    <li>Company: Biotium</li>
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    <li>REF: 41003</li>
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    <li><a href="https://biotium.com/product/gelredtm-nucleic-acid-gel-stain-10000x-in-water/"><p>for further information click here</p></a></li>
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<h3>Anti-Human-AnnexinV</h3>
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    <li>Product: anti-human Annexin V/Biotin </li>
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    <li>Company: Affymetrix eBioscience</li>
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    <li>REF: BMS147</li>
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    <li><a href="http://www.ebioscience.com/human-annexin-v-antibody-biotin-vaa-33.htm"><p>for further information click here</p></a></li>
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<h3>Gel Loading Dye</h3>
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    <li>Product: Gel Loading Dye, Purple (6X), no SDS </li>
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    <li>Company: New England BioLabs</li>
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    <li>REF: B7025S</li>
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    <li><a href="https://www.neb.com/products/b7025-gel-loading-dye-purple-6x-no-sds"><p>for further information click here</p></a></li>
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<h3>T4 DNA Ligase</h3>
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<ul>
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    <li>Product: T4 DNA Ligase</li>
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    <li>Company: New England BioLabs</li>
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    <li>REF: M0202S</li>
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    <li><a href="https://www.neb.com/products/m0202-t4-dna-ligase"><p>for further information click here</p></a></li>
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<h3>PstI</h3>
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    <li>Product: PstI</li>
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    <li>Company: New England BioLabs</li>
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    <li>REF: M0140S</li>
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    <li><a href="https://www.neb.com/products/r0140-psti"><p>for further information click here</p></a></li>
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<h3>DNA Ligase Buffer</h3>
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<ul>
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    <li>Product: Taq DNA Ligase Reaction Buffer </li>
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    <li>Company: New England BioLabs</li>
 +
    <li>REF: B0208S</li>
 +
    <li><a href="https://www.neb.com/products/b0208-taq-dna-ligase-reaction-buffer"><p>for further information click here</p></a></li>
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Revision as of 15:04, 8 September 2015

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

Materials

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 and Kits

LB-Broth

Agarose

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

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).

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).

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.

SK-BR-3

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

Machines

Nano Drop

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

    • for further information click here

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Plate Reader

Absorbance Measurments

UV- and Dark Hood

Large Centrifuge

Small Centrifuge

Shaker

PCR Machine

Gel Electrophoresis

Eppi-shaker/Heatingblock

Vortex

Balance

Incubator

FACS

Fluorescence Scanner for Western Blots