Difference between revisions of "Team:ETH Zurich/Materials"
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Revision as of 12:57, 7 September 2015
- Project
- Modeling
- Lab
- Human
Practices - Parts
- About Us
Materials
Buffers and media
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 )
Chemicals and 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).
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