Team:UCL/Project/Protocols

5X M9 Salts

In 500mL Duran bottle insert:

Minimal Agar

Mix:

Generating Competence Cells

Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.

Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.

After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).

Streaking Plates

Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight.

Glycerol Stock Generation

12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).

Transformation

1) Remove one of your aliquots of competent cells from the -80C freezer and place onto ice. 2) Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes. 3) Place tubes into 37C water bath for 10 minutes (heat shock). 4) Place tubes into ice for 2 minutes. 5) Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C. 6) Centrifuge at high RPM for 2 minutes. Discard the supernatant. 7) Resuspend cell pellet into 100uL of LB. 8) Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity. 9) Incubate for 16 hours at 37C and then pick colonies if growth has occurred.

Analytical Digest

Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:

Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest.

Nanodrop

Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.

Ligation

In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume

Passaging Adherent Cells

In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood.

1) Pipette spent medium and discard to waste.

2) Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.

3) Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C.

Stable Transfection Of Adherent Cells

For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.

1) The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.

2) Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.

3) Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.

Amyloid Degradation Assay

Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.

To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.

Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.

To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.

Congo Red Spectrophotometric assay

1) Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.

2) At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.

3) To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.

4) Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.