Revision as of 13:06, 7 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.

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