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

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<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>  
 
<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>  
  
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<h3>3T3 P65 -/- </h3>
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<p>Murine fluorescent fibroblast cell line.
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<ul>
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<li> H2B-GFP: nuclear staining</li>
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<li> P65- dsRed: cytoskeleton</li>
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</ul>
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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>
  

Revision as of 15:38, 17 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. These cells were provided by the Panke Group at ETH Zurich.

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). 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
  • 1M 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

Fluorescence Scanner for Western Blots

Incubator

Large Centrifuge

Nano Drop

PCR Machine

Plate Reader

Shaker

Small Centrifuge

UV- and Dark Hood

Vortex

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