Difference between revisions of "Team:ETH Zurich/Materials"
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<h2>Buffers and media </h2> | <h2>Buffers and media </h2> | ||
+ | |||
<div class="info"> | <div class="info"> | ||
− | <h3> | + | <h3>Annexin V binding buffer</h3> |
<ul> | <ul> | ||
− | <li> | + | <li>10 mM HEPES</li> |
− | <li> | + | <li>140 mM NaCl</li> |
− | <li> | + | <li>2.5 mM</li> |
− | <li> | + | <li>250 mM CaCl<sub>2</sub></li> |
− | <li> | + | <li>pH 7.4</li> |
+ | <li>Filter the solution through a 0.2 µm filter. </li> | ||
</ul> | </ul> | ||
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</div> | </div> | ||
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<div class="info"> | <div class="info"> | ||
− | + | <h3>Jurkat medium</h3> | |
− | <h3> | + | |
<ul> | <ul> | ||
− | <li> | + | <li>2 mL MEM 1x</li> |
− | <li> | + | <li>20 mL FBS 100%</li> |
− | <li | + | <li>2 mL GlutaMax 100x</li> |
− | <li> | + | <li>0.4 mL Penicillin (10k units/mL) /Streptomyocin (10k µg/mL )</li> |
+ | <li>200 mL RPMI</li> | ||
</ul> | </ul> | ||
− | |||
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− | + | <h3>TFP1</h3> | |
− | <h3> | + | |
<ul> | <ul> | ||
− | <li> | + | <li>100 mM RbCl</li> |
− | <li>50 | + | <li>50 mM MnCl2</li> |
− | <li> | + | <li>30 mM potassium acetate</li> |
− | <li> | + | <li>10 mM CaCl<sub>2</sub></li> |
+ | <li>15%glycerol</li> | ||
</ul> | </ul> | ||
− | |||
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− | <h3> | + | |
+ | <h3>TFP2</h3> | ||
<ul> | <ul> | ||
− | <li> | + | <li>100 mM MOPS</li> |
− | <li> | + | <li>50 mM RbCl</li> |
− | <li>2 | + | <li>75 mM CaCl<sub>2</sub></li> |
− | + | <li>15%glycerol</li> | |
− | <li> | + | |
</ul> | </ul> | ||
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− | <h3> | + | <h3>3T3 medium</h3> |
<ul> | <ul> | ||
− | <li> | + | <li>500 mL D-MEM 1x</li> |
− | <li> | + | <li>50 mL FBS 100%</li> |
− | <li> | + | <li>5 mL GlutaMax 100x</li> |
− | <li> | + | <li>5 mL Penicillin (10k units/mL) /Streptomyocin (10k µg/mL )</li> |
− | + | ||
− | + | ||
</ul> | </ul> | ||
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Revision as of 16:07, 8 September 2015
- Project
- Modeling
- Lab
- Human
Practices - Parts
- About Us
Materials
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).
SK-BR-3
HER2-positive breast cancer cell line. We used this cell line to validate the chimeric receptors of the Stockholm iGEM team.
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.
Machines
Emulsifier
- EmulsiFlex-C3 from AVESTIN, Inc. and AVESTIN Europe GmbH/li>
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Fluorescence Scanner for Western Blots
- ODYSSEY® CLx Infrared Imaging System
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Nano Drop
- Thermo Scientific NanoDrop® 2000 UV-Vis spectrophotometer combined with NanoDrop 2000 / 2000c Software
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Plate Reader
- Tecan Infinite M200 Pro™ combined with Tecan iControl TM software
for further information (platereader) click here
for further information (software) click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
UV- and Dark Hood
- Biostep Dark-Hood DH-50™ combined with Argus-X1™ software and Biostep argusX1™ basic licence
for further information (hood) click here
for further information (software) click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
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.
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
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
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Chemicals
LB-Broth
- Product name: BD Difco™ LB Broth, Miller (Luria-Bertani)
- Company: BD
- REF: 244620
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Agarose
- Product name: Agarose
- Company: Sigma-Aldrich
- REF: A9539-500G
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Agar
- Product name: Agar für die Bakteriologie BC
- Company: Sigma-Aldrich
- REF: A0949
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Glycerol
- Product: Glycerol anhydrous BioChemica BC
- Company: AppliChem
- REF: A1123
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
2-log DNA ladder
- Product: 2-Log DNA Ladder (0.1-10.0 kb)
- Company: New England BioLabs
- REF: N3200S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Taq DNA Ligase
- Product: Taq DNA Ligase
- Company: New England BioLabs
- REF: M0208S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Cutsmart Buffer
- Product: CutSmart® Buffer
- Company: New England BioLabs
- REF: B7204S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
EcoRI
- Product: EcoRI-HF®
- Company: New England BioLabs
- REF: R3101S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Q5 Master Mix
- Product: Q5® High-Fidelity 2X Master Mix
- Company: New England BioLabs
- REF: M0492S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
dNTP Mix
- Product: Deoxynucleotide (dNTP) Solution Mix
- Company: New England BioLabs
- REF: N1201A
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Taq DNA Polymerase
- Product: Taq DNA Polymerase with ThermoPol® Buffer
- Company: New England BioLabs
- REF: M0276L
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
AnnexinV-Alexa
- Product: Annexin V, Alexa Fluor® 488 conjugate
- Company: ThermoFischer Scientific
- REF: A13201
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Streptavidin Beads
- Product: Roti®-MagBeads Streptavidin
- Company: Carl Roth
- REF:
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Red Gel Stain
- Product: GelRed Nucleic Acid Gel Stain, 10,000X in water
- Company: Biotium
- REF: 41003
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Anti-Human-AnnexinV
- Product: anti-human Annexin V/Biotin
- Company: Affymetrix eBioscience
- REF: BMS147
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Gel Loading Dye
- Product: Gel Loading Dye, Purple (6X), no SDS
- Company: New England BioLabs
- REF: B7025S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
T4 DNA Ligase
- Product: T4 DNA Ligase
- Company: New England BioLabs
- REF: M0202S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
DNA Ligase Buffer
- Product: Taq DNA Ligase Reaction Buffer
- Company: New England BioLabs
- REF: B0208S
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
DMEM
- Product: DMEM, high glucose, no glutamine, no phenol red
- Company: Gibco ThermoFisher Scientific
- REF: 31053-028
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
DMEM with Glutamine
- Product: DMEM cell culture media
- Company: Gibco ThermoFisher Scientific
- REF: 41965-039
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
RPMI
- Product: RPMI 1640 Medium, no phenol red
- Company: Gibco ThermoFisher Scientific
- REF: 11835-063
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
Accutase
- Product: StemPro® Accutase® Cell Dissociation Reagent
- Company: Gibco ThermoFisher Scientific
- REF: A11105-01
for further information click here
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
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.
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
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.
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)
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).
![](https://static.igem.org/mediawiki/2015/1/1f/Blank_square.png)