"What I cannot create I do not understand."
- Richard Feynmann

Experiments & Protocols

Introduction

In the course of our project we performed a lot of different experiments, ranging from construction and transformations of plasmids into E. Coli over the testing their functionality and response measurements after induction of our circuit, up to experiments with mammalian cells which are the subjects of our analysis.

Below you can find the protocols for all our experiments.

Buffers and mediums

TFP1

100 mM RbCl

50 mM MnCl2

30mM potassium acetate

10mM CaCl₂

15%glycerol

TFP2

100 mM MOPS

50 mM RbCl

75 mM CaCl₂

15%glycerol

SOC medium

Tryptophane 20g/L

Yeast extract 5g/L

NaCl 0.5g/L

250mM KCl

1M MgCl₂

50% (w/v) sterile glucose

Filter the solution through a 0.2 µm filter.

3T3 medium

D-MEM 1x: 500 mL

FBS 100%: 50 mL

GlutaMax 100x: 5 mL

Penicillin (10k units/mL) /Streptomyocin (10k µg/mL ): 5 mL

Jurkat medium

MEM 1x: 2 mL

FBS 100%: 20 mL

GlutaMax 100x: 2 mL

Penicillin (10k units/mL) /Streptomyocin (10k µg/mL ): 0.4 mL

RPMI: 200 mL

General useful things

Cryostock of bacteria

Add 1 mL of 40% glycerol to a cryogenic vial.
Add 1 mL sample from the bacterial culture.
Vortex the vial.
Store in -80ºC freezer.

Preparation of chemically competent cells

Take a trace of Escherichia coli from a glycerol stock vial and put it on a LB plate with the correspondent antibiotic.
Incubate overnight at 37ºC.
Pick a colony and inoculate 10mL LB medium containing the relevant antibiotic. Grow overnight at 37ºC.
Add 1mL overnight culture to 100mL prewarmed LB medium containing the relevant antibiotic in a 500mL flask, and shake at 37ºC until an OD₆₀₀ of 0.5 is reached (usually 90-120 min).
Cool the culture on ice 5min, and transfer the culture to a sterile, round-bottom centrifuge tube.
Collect the cells by centrifugation at low speed (5 min, 4000 xg, 4ºC).
Resuspend the cells gently in cold (4ºC) TFB1 buffer (30 mL for 100 mL culture) and keep the suspension on ice for 90min.
Collect the cells by centrifugation (5min, 4000 xg, 4ºC).
Discard the supernatant carefully. Always keep the cells on ice.
Resuspend the cells carefully in 4 mL ice-cold TFB2 buffer
Prepare aliquots of 100-200µL in sterile microcentrifuge tubes and freeze in dry-ice-ethanol mix. Store the competent cells at -70ºC

Transformation of chemically competent cells

Take 1 µL DNA to be transformed and put into a cold sterile 1.5 mL microcentrifuge tube, and keep on ice.
Thaw an aliquot of frozen competent cells on ice.
Resuspend the cells and transfer 50 of the cell suspension into the microcentrifuge tube with the plasmid DNA, mix carefullu, and keep on ice for 20 min.
Transfer the tube to a 42ºC water bath for 90 s.
Add 500 µL SOC medium to the cells and incubate for 60-90 min at 37ºC. Shaking increases transformation efficiency.
Plate 100 µL on LB-agar plates containing antibiotics. Incubate the plates overnight at 37ºC.

Team:ETH Zurich/Experiments