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

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<h3>TOP10</h3>
 
<h3>TOP10</h3>
 
<p>The bacterial strain we used as a base for all our transformations was TOP10.</p>
 
<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>
 
<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).</p>
  
 
<h3>HL60</h3>
 
<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).</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>
  
 
<h3>SK-BR-3</h3>
 
<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>

Revision as of 14:12, 6 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

  • Product name: Difco™ LB Broth, Miller (Lurica-Bertani)
  • Company: BD
  • Lot: 4339957
  • CAS:

Agarose

  • Product name: Agarose
  • Company: Sigma-Aldrich
  • Lot: #SLBK3392V
  • CAS: 9012-36-6

Glycerol

  • Product: Glycerol anhydrous BioChemica
  • Company: AppliChem
  • Lot:
  • CAS:

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.

Put reactions

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

Plate reader

Tecan Infinite M200 Pro™ combined with Tecan iControl TM software

Absorbance measurments

Novaspec Plus™ Visible Spectrophotometer

UV- and dark hood

Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence

Large Centrifuge

Eppendorf Centrifuge 5810 R

Small Centrifuge

Eppendorf Centrifuge 5424 R

Shaker

Kuhner Climo-Shaker ISF1-X

PCR machine

Applied Biosystems Veriti 96Well Thermal Cycler

Gel electrophoresis

Helixx Technologies MyRun®

Eppi-shaker/Heatingblock

Eppendorf Thermomixer compact

Vortex

Scientific Industries VortexGenie®2

Balance

MettlerToledo XS4002S DeltaRange® Balance

Incubator

Thermo Scientific Heraeus Incubator

FACS

Sterile hood