How to make LB medium?

Recipe for 500 ml LB medium:

• 5 g Tripton
• 2.5 g NaCl
• 2.5 g Yeast extract
• 500 mL MQ water
• glass bottle

Steps:

After you have measured the ingredients, mix the solution with a magnetic stirrer approx. 10 minutes or until the components dissolve.
You should sterilize them in an autoclave and after they have cooled down they are ready to use. Add the right antibiotics to the medium in 1:1000 dilution.

How to pour agar plates?

• Prepare your LB agar medium.
• If you autoclaved it freshly please wait until it cooled down enough to handle. (<60°C)
• If you work with a previously prepared medium, you must warm it up with a microwave oven.
• Please loosen the cap of the bottle.
• Warm it for about 1 minute, and shake it vigorously.
• Then continue the warming up but with close attention. You should mix it every 20-30 seconds or if you see intense bubbling.
• You can stop when the solution is clear and you do not see any gel floating.
• You have to wait until it's cool enough to handle with your bare hands.
• From this step continue your work in a sterile environment!
• Place your plates in a laminar box.
• When you are opening your bottle, sterilize the opening with flame.
• Add antibiotics at a 1:1000 ratio.
• Pour as fast as possible approx. 8-10 mL medium into the plate, close it, and mix it with a circular motion.
• Wait for 20 minutes to solidify.
• Store the plates at 4°C.

Insert restriction digestion and purification

• Weigh the reaction mix in an Eppendorf:
• Add the required enzymes, the DNA template, and the right buffer (buffers were chosen according to Thermo’s webpage, DoubleDigest).
• Restriction enzymes used for construct digestion:
• J23101-BLADE-mCherry: XbaI, XhoI
• J23101-BLADE-ClyA: XbaI, XhoI
• J23101-ClyA: XbaI, XhoI
• J23101-Intimin-EGFR: XbaI, PaeI
• J23101-Intimin-CEA: XbaI, PaeI
• J23101-sfGFP: XhoI, PaeI
• Put it in a 37°C incubator for one hour or overnight.
• Purification according to the kit protocol.

We performed DNA purification with a nucleic acid purification kit (Thermo Fischer Scientific: GeneJET PCR Purification Kit; https://www.thermofisher.com/order/catalog/product/K0701) according to the manufacturer's protocol (differences: elution volume: 25 µL).

Plasmid digestion

• Add the required enzymes, the DNA template, and the buffer.
• Restriction enzymes used for plasmid digestion:
• pET28a: XbaI, XhoI
• pEV: XbaI, XhoI
• pETARA: XbaI, XhoI
• Put it in a 37°C incubator for one hour or overnight.
• Mix the reactions with 6x Purple Loading Dye.
• Add Generuler 1 kbp Plus Marker. (Thermo: https://www.thermofisher.com/order/catalog/product/SM1331)
• Run agarose gel electrophoresis, 100 mV, 45 min.
• Cut out the band of digested plasmid with a sterile scalpel.
• Purification according to the kit protocol. (Thermo Fischer Scientific: GeneJET Agarose Purification Kit: https://www.thermofisher.com/order/catalog/product/K0691)

Ligation

• Calculate how many µL of insert and vector you need to introduce to the reaction mix based on the equitation below:
  required mass insert (g) = desired insert/vector molar ratio x mass of vector (g) x ratio of insert to vector lengths
• Add T4 ligase, 10x Ligase buffer, sterile distilled water, and DNA components in appropriate proportions.
• Incubate at room temperature for 1 hour.

• Add 10x T4 DNA Ligase reaction buffer, calculated insert and vector ng-s, sterile distilled water, Hi-T4 ligase.
• Incubation for 10 minutes at 37°C, heat inactivation for 10 minutes.

Transformation

• Competent cell name: DH5α
• Competent cell volume: 100 µL
• Plasmid vector volume: 1 µL
• Mix the plasmid with cells
• Incubation on ice: 1 min
• Heat shock: temperature: 42°C, time: 2 min
• Incubation on ice: 5 min
• LB medium volume: 0.1 mL add to transformation mix
• LB + Antibiotic plate: Pipette the cells onto the agar and then spread them out with the burnt and cooled metal stick. Smear the cells until they are absorbed by the agar. Seal the edge of the plate with cork film and place it in the incubator.
• Incubation: temperature 37°C, time: overnight


• Competent cell name: Xl1Blue
• Competent cell volume: 40 µL
• Plasmid vector volume: 1 µL
• KCM mix: 8 µL
• Sterile distilled water: 27 µL
• Mix the plasmid with cells
• Incubation on ice: 20 min
• Heat shock: room temperature, 10 min
• LB medium volume: 0.1 mL add to transformation mix, shake at 180 rpm at 37°C, 1 hour.
• LB + Antibiotic plate: Pipette the cells onto the agar and then spread them out with the burnt and cooled metal stick. Smear the cells until they are absorbed by the agar. Seal the edge of the plate with cork film and place it in the incubator.
• Incubation: temperature 37°C, time: overnight

Growing bacteria

• Pick up colonies with a pipette tip and put them in 5 mL of LB-medium with 1000x antibiotics (ampicillin/carbenicillin)
• Then incubate at 37°C O/V, shaking at 180 rpm.

Miniprep

Isolation of plasmid from overnight culture according to the kit protocol (we selected Thermo Fischer Scientific:GeneJET Plasmid Miniprep Kit:
https://www.thermofisher.com/order/catalog/product/K0502).
After the purification was measured the concentration with NanoDrop.

Diagnostic Digestion

• Weigh the reaction mix in an Eppendorf.
• Add the required enzymes, the DNA template, and the selected buffer (for each enzyme pair we selected the appropriate buffers according to Thermo Fisher DoubleDigest web page).
• Incubate at 37°C for 1 hour.
• Mix the reactions with 6x Purple Dye.
• Add Generuler 1 kbp Plus Marker. (Thermo,
  https://www.thermofisher.com/order/catalog/product/SM1331)
• Run agarose gel electrophoresis, 100 mV, 45 min.

Gel run

• Measure 0.5 g agarose on the scale, pour it into a flask and fill it with 50 mL of 1x TAE buffer.
• This mixture is heated in a microwave oven to obtain a syrupy liquid without translucent agarose particles (this can be achieved by shaking the jar frequently while wearing protective gloves).
• When the solution is ready, cool the flask to hand temperature and add EcoSafe 2.5 µL (from 4°C) of dye to the agarose.
• The mixture is poured into the bath and any bubbles that may form are to be destroyed with a pipette tip.
• Leave to solidify for at least 20 minutes. After the gel has solidified, the vat is filled with 1x TAE buffer to cover the gel by half a cm.
• Run gel electrophoresis at 100 mV, for 45 min.

BL21 transformation

For one construction:

• 40 µL BL21 competent cells
• 8 µL KCM mix (calcium and magnesium ion mix)
• 27 µL sterile distilled water
• 1 µL plasmid
• 20 min on ice
• 10 min room temperature
• Add 100 µL LB-medium.
• Shake on ~180 RPM, 37°C, for at least 1 hour.
• Add the mix to LB-medium for starter culture (usually 50 mL).
• Add 1:1000 antibiotics.

Making competent cells

Storage solutions should be stored in sterile containers at 4°C. 100 mL should be made at a time.

• 100 mM RbCl
• 50 mM MnC
• 30 mM Potassium acetate
• 10 mM CaCl₂
• 15 % glycerol

TFB solution II: pH=6.8 (adjust carefully with KOH)

• 10 mM MOPS
• 10 mM RbCl
• 75 mM CaCl₂
• 15 % glycerol

Pre-culture:

• Dip sterile tip (under sterile booth) into frozen BL21 cells (-70°C).
• Make 5 mL LB with selection antibiotics + tip with the cells (pipette tip should also be placed in the vial).
• Keep it in a shaker overnight at 37°C, 200 rpm.

Culture:

• Make a 50 mL LB culture medium with antibiotics from 500 µL BL21 (this is the pre-culture grown the night before).
• Keep the cultures for 120 minutes at 37°C (afterward 1 mL sample should be taken at intervals, optical density is measured at 600 nm when OD is between 0.3-0.6 (≈0.4), and the solution can be removed from the shaker.
• Transfer 50 mL to a sterile falcon and centrifuge at 3600 rpm, 20 minutes at 4°C.
• Discard supernatant.
• Resuspend the cells in 10 mL TFB I.
• Keep it for 20 minutes on ice.
• Keep it for 20 minutes at 3600 rpm, 4°C.
• Discard supernatant.
• Resuspend the cells in 1 mL TFB II.
• Keep it for 20 minutes on ice.

Freezing:

• After the addition of 15 % sterile filtered glycerol (regardless of whether competent preparation solutions already contain glycerol).
• Put in it 1 mL 350 µL 50% glycerol.
• Put 80-100 µL from it into Eppendorf tubes
• Store at 80°C.

Cell culturing

Materials:

• Complete medium
• Serum-free DMEM (Dulbecco’s Modified Eagle Medium, Gibco, Thermo Fisher Scientific, Waltham, MA, USA)
• 10 % FBS
• 5 mM glutamine
• 1 % penicillin-streptomycin
• PBS
• Trypsin
• Resasurin (Thermo Fischer)
• Sterile Flasks (T-25, T-75)
• Serologic pipettes

Thawing cells

• Frozen cells are stored in liquid nitrogen until immediate use. When removing from liquid nitrogen, place vials on dry ice for a few minutes, or start thawing immediately.
• The amount in 1 cryovial (1 mL) is sufficient to start a culture in a 25 cm² flask.

Work quickly and carefully.

Prepare

• Complete medium pre-heated in an incubator (37°C, 5 % CO₂) in the flask to be used for culturing (25 cm² flask with 5-6 mL cell culture media). It is important to avoid excessive alkalinity of the medium during the recovery of the cells. It is suggested that, before the addition of the vial contents, the culture vessel containing the complete growth medium be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
• Complete medium pre-heated
• Pre-heated water bath (37°C)
• 5 mL pipettes (sprayed with EtOH)
• 15 mL tubes (sprayed with EtOH)

Steps

• Remove the vial of frozen cells from liquid nitrogen and quickly thaw in a 37°C water bath by gentle agitation (it should take between 1 and 2 min). To reduce the possibility of contamination, keep the O-ring and cap out of the water. Remove from the water bath when there is only a small ice chunk inside the vial (it will thaw by the time you start the process).
• Just before the cells are completely thawed, decontaminate the outside of the vial with 70 % EtOH and transfer the cells to a sterile 15 mL tube.
• Add 9 mL of pre-heated medium to the cells. Start very carefully dropwise then you can add faster.
• Centrifuge the cells at 500 x g for 5 minutes at room temperature.
• Remove the medium using a pipette very carefully.
• Resuspend the cell pellet in the pre-heated media for the cell culture flask.
• Place the cell suspension into the cell culture flask and place it in an incubator (37°C, 5 % CO₂).
• Incubate overnight or for two days.

Note: Wait for a few passages (2-3) until cells are fully recovered before starting to use them.

Subculturing

• Passage cells when they are 80 % confluent.
• Passage cells 2-3 times a week.

ALWAYS label the flask: the date, your name, passage number, cell type, and subculturing ratio.

• Remove supernatant with a pipettor.
• Pipette 5 mL of preheated PBS, and wash out the remnant medium. Wash the cells gently but thoroughly.
• Trypsinize cells with 1 mL 0.25 % trypsin solution in the case of the T25 flask (in the case of T75 use 2 mL).
• Incubate A431 cells for 15 min at 37°C, 5 % CO₂.
• Incubate HTC116 cells for 2-3 min at 37°C, 5 % CO₂.
• In the meantime prepare a flask with 4 mL media and incubate it for at least 15 min at 37°C, with 5 % CO₂.
• Add 2 mL of pre-heated media to stop the enzyme reactions.
• Hit gently the bottom of the flask a few times.
• Check the detachment of the cells.
• Suspend cells with a pipettor, and place the cell suspension into a clean Falcon tube.
• Centrifuge cells for 5 min at 500 g.
• Remove supernatant and resuspend cells in 5 mL of pre-heated media.
• Add 1 mL cell suspension to the pre-heated flask.
• Check cell density.

Cell counting

• Clean the chamber and cover the slip with alcohol. Dry and fix the coverslip in position.
• Harvest the cells.
• Add 10 μL of the cells to the hemacytometer. Do not overfill.
• Place the chamber in the inverted microscope under a 10X objective.
• Count the cells in the large, central gridded square (1 mm²). The gridded square is circled in the graphic below. Multiply by 10^4 to estimate the number of cells per mL. Prepare duplicate samples and average the count.

Cell freezing

Freeze cells at a density of at least 1 x 10^6 cells/mL (total volume in 1 vial: 1 mL)
(A T-75 flask usually yields 3-4 cryovials.)

Prepare:

• Ice bucket
• Labeled cryovials (2 mL)
• Mr. Frosty (filled with IPA and pre-chilled on ice or a few minutes at -80°C)
• Liquid nitrogen container for cryovials

Steps:

• Culture the desired quantity of cells to 70-90 % confluency. (T-75 flask).
• Remove all medium from the flask and wash the cells once with 10 mL PBS to remove excess medium and serum. The serum contains inhibitors of trypsin.
• Add 2 mL trypsin solution to the monolayer and incubate the cells for the time given above, until the cells detach.
• Add 10 mL complete medium and transfer the cell suspension to a 15 mL sterile conical tube.
• Count cells.
• Place the right amount of cell suspension into the cryovial.
• Total it for 0,9 mL with media.
• Supplement the solution with 100 µL DMSO.
• Place the vials (max 18) in Mr. Frosty compartments. Place Mr. Frosty at -80°C for at least 24 h. (Mr. Frosty is a controlled-rate freezing apparatus ensuring a freezing rate of a decrease of 1°C per minute).
• Transfer cryovials to liquid nitrogen storage.

Cell viability assay

• Seed cells into 96-well plate (in antibiotic-free DMEM).
• After 24 h remove the supernatant and add 100 µL 1xPBS and resazurin dye solution in an amount equal to 10 % of 1xPBS volume.
• Return cultures to the incubator for 2 h.
• Read fluorescence at a wavelength of 590 nm using an excitation wavelength of 560 nm (SpectraMax ID3 Multi-Mode Microplate Reader, Molecular Devices).

SDS-PAGE

First day: