In the lab, we used different experimental procedures. There are protocols for the wet and bacterial lab, for the cell culture lab and for the electrophysiology lab.
Cell Culture
Medium of HEK 293 cells
Medium contents
DMEM (Gibco N° 41965-039) 500 ml
50 ml (10 %) FCS = (Hyclone)
10 ml Penicillin/Streptomycin (Gibco N° 15070-071) = 100-100U/ml
10 ml Glutamax (Gibco N° 35050-038) = 10 mM
5 ml Non-Essential-Amino-Acids (Gibco N° 1140-035) = 10x
Growing Conditions
Incubate at 37°C and 10% CO2
Split cells around 70-90% confluency
Change medium twice a week
Standard procedure for trypsinization
Wash the cells with Versene (Gibco N° 15040-033, ± 2 min)
Remove versene and add Trypsin (Gibco N° 25300-096)
Incubate at 37°C (around 3-5 min)
Stop the trypsinization by adding 5-10 times volume of medium
Count cells
Centrifuge for 5 minutes at 1200 rpm
Aspirate supernatant and resuspend the cells in medium
Plate cells for experiments or culture
Coating of glass coverslips (for patch-clamp or Ca2+ imaging)
Poly-L-lysine (PLL, sigma P2636)
Dissolved at 0.1 mg/ml in Milli-Q H2O
Stored at –20°C
Once opened stored at RT
Coverslips (18mm)
Prepare 12-well plates with a coverslip in each well
Transfer 1 sterilized coverslip to each well
Add 350 ml of PLL to each coverslip
Incubate for 20 minutes at RT
Aspirate and add Milli-Q H2O to each coverslip
Incubate for 20 minutes at RT
Aspirate H2O and store for experiments. The plates can be kept for several days at 4°C.
Seeding Cells
Seeding cells for single-cell experiments
Spray your hands with ethanol, take the 6 well plate with cells out of the incubator and check the density of the cells
Place the cells under the flow
Remove the medium
Wash the cells with 1 ml of versene for 2 minutes
Remove versene and add 500μl of trypsin
Incubate at 37°C, 10% CO2 for about 5-10 minutes
Stop the trypsinization by adding 5 ml of medium.
Transfer cell suspension to a 50 ml Falcon tube
Separate the cells by pulling them with a 5 ml syringe through a black needle of 22G (5-10 times)
I. Ca2+ imaging
Cells are typically seeded in a 12-well plate with PLL coated round coverslips of 18 mm
Add 1 ml of medium to each 12-well of your plate
If the cells in the 6-well had a density of 95-100%, typically seed 400-500 microliters of cell suspension per 12-well (density depending on experimental needs)
Incubate at least 2 hours for cell attachment to the coverslip
II. Patch clamp
Cells are typically seeded in a 12-well plate, on PLL coated round coverslips of 18 mm
Add 1 ml of medium to each 12-well of your plate
If the cells in the 6-well had a density of 95-100%, typically seed 100-300 microliters of cells per 12-well (density depending on experimental needs)
Incubate at least 2 hours for cell attachment to the coverslip
Ca2+ imaging
Fura-2, AM (MW: 1001,9)
Dilute 50 μg of Fura-2 in 50 μl DMSO (1 mM stock solution)
Make aliquots of 2 μl in brown eppies and store at -20°C
To load cells (1 well): add 2 μl of Fura-2 to 1 ml of medium (2μM work solution)
Incubate cells for 30 minutes before measurement
Wet Lab
Transformation of MAX Efficiency™ DH5α™ Competent Cells
Day 1
Add 25µL of bacteria to the bottom of a polypropylene 14mL Falcon tube (352059). Place on ice.
Add either 0.5µL of 100ng/µL plasmid or 1 to 5µL of a ligation mix to the bacteria. Mix gently by shaking. Leave on ice for 30 minutes
Preheat the SOC medium and waterbath to 42ºC.
Perform a heat shock. Place cells at 42ºC for 30 to 45 seconds. Return to ice for 2 minutes.
Add 0.5mL SOC medium.
Place in shaking incubator at 37ºC for 1 hour.
Plate and spread on plates containing 50µg/mL of a specific antibiotic resistance markers. Incubate overnight at 37ºC for maximum 16 hours.
Day 2
Add 3mL of LB medium containing 50µg/mL of a specific antibiotic resistance marker to a 14mL polystyrene Falcon tube (352057).
Pick a single colony using a Inoculation loop. Transfer colony into medium and shake. In order to perform a miniprep, grow at 37ºC overnight and skip to 'Day 3'. Otherwise, grow for 3 to 4 hours and continue.
Add 70mL LB medium containing 50µg/mL of a specific antibiotic resistance marker to a sterile erlenmeyer.
Add the bacteria to the new medium: ½ of the medium in the Falcon if the growth is visible, all of the medium if the solution is still clear.
Place erlenmeyers in shaking incubator at 37ºC overnight or for maximum 16 hours.
Day 3
Harvest plasmids using mini-, midi- or maxiprep kit.
