Experiments

Lab protocols

Below, the protocols for all wet lab steps can be found.

LAMP p53

The recognition sequence is a p53 sequence taken from Marciniak et al. 2008 (see: parts), that we have added to our aptamer construct via a linker, and that is recognized by the padlock probe. We ran RCA-LAMP on the recognition sequence alone first to make sure that LAMP worked. RCA-LAMP consists of three main parts: phosphorylation of padlock probe, preparation of ligation mix, preparation of amplification mix.

Preparations

  • Sterile water: Pour 250 mL deionized water into a 500 mL flask and autoclave it.
  • 24 uM FIB primer: 10 uL of 100 uM FIB (stock solution) with 32 uL of water.
  • 24 uM BIP primer: 10 uL of 100 uM BIP (stock solution) with 32 uL of water.
  • 2.5 uM DNA sequence (aptamer/recognition sequence): 2 uL of 100 uM of DNA with 78 uL of water.

Protocol

  1. Phosphorylation of padlock probe:
    1. Add in an eppendorf tube 3 uL of padlock probe.
    2. Add 5 uL of 10x T4 DNA ligase buffer.
    3. Add 1 uL of T4 polynucleotide kinase.
    4. Add 41 uL of water to make a 50 uL solution.
    5. Incubate for 30 min at 37°C, then for 20 min at 65°C.
  2. Ligation mix:
    1. Add in an eppendorf tube 1 uL of 10x T4 DNA ligase buffer.
    2. Add 1 uL of diluted DNA sequence.
    3. Add 1.7 uL of phosphorylated linear padlock probe.
    4. Add 1 uL of T4 DNA ligase.
    5. Add 5.3 uL of water to make 10 uL mix.
    6. Incubate for 1 hour at room temperature.
    7. PCR DNA cleanup from New England Biolabs to clean the DNA in ligation mix from any impurities.
      1. Add 70 uL of binding cleanup buffer to the eppendorf tube.
      2. Transfer the 80 uL solution into a column and insert the column in a collection tube.
      3. Centrifuge the tube for 1 min. Discard flow-through and reinsert column into collection tube.
      4. Add 200 uL of wash buffer in column and centrifuge for 1 min. Discard is optional.
      5. Repeat the previous step. Discard flow-through and collection tube.
      6. Insert column into an eppendorf tube and add 6 uL of elution buffer. Incubate for 1 min before centrifuging. Discard column.
  3. Amplification mix:
    1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
    2. Add 2 uL of diluted FIB and 2 uL of diluted BIP.
    3. Add 6 uL of water.
    4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 h at 65°C.

LAMP - Magnetic Beads

After monitoring that RCA-LAMP was successful, the next step was to see whether the aptamer would detect tau protein. To capture tau, we used magnetic nickel beads. Tau binds to the beads thanks to the fusion of a his-tag to the tau protein. Aptamers were added to tau in beads and then LAMP was performed.

Preparations

  • Equilibration buffer: Make 1 L of equilibration buffer by adding 6,03 g of 50mM Tris, 1,36 g of 20 mM Imidazole and 17,53 g of 300 mM NaCl and then filling up the flask until the 1 L mark is reached. The elution buffer should have a pH of 8. Adjust pH if necessary.
  • Elution buffer: Make 0,5 L of elution buffer by adding 3,02 g of 50 mM Tris, 17,02 g of 20 mM Imidazole and 8,77 g of 300 mM NaCl and then filling up the flask until the 500 mL mark is reached. The elution buffer should have a pH of 7. Adjust pH if necessary.
  • Sterile water: Pour 250 ml deionized water into a 500 ml flask and autoclave it.
  • A PCR DNA cleanup kit is needed.

Protocol

  1. Tau binding to magnetic beads:
    1. Resuspend magnetic beads by vortexing and immediately pipette 50 uL into an eppendorf tube.
    2. Add 100 uL of dialysed tau and place the tube on an end-over-end shaker for 1 h.
    3. Place the tube on a magnetic rack and wait for 1 min before pipetting out and discarding supernatant.
    4. Wash beads with 500 uL of equilibration buffer (20 mM of imidazole). Add the buffer, place the tube in the magnetic rack for 1 min and discard the buffer.
    5. Add 1 uL of 100 uM aptamer to the beads and dilute with 200 uL of equilibration buffer.
    6. Place the tube on an end-over-end shaker for 1 h, then place it on the magnetic rack for 1 min.
    7. Discard the supernatant and wash with 500 uL equilibration buffer.
    8. Add 50 uL of elution buffer (200 uM imidazole), incubate for 1 min, place on magnetic rack for 1 min and collect eluate.
  2. Ligation mix:
    1. Add in an eppendorf tube 1 uL of 10x T4 DNA ligase buffer.
    2. Add 5 uL of eluate.
    3. Add 1.7 uL of phosphorylated linear padlock probe.
    4. Add 1 uL of T4 DNA ligase.
    5. Add 1.3 uL of water to make 10 uL mix.
    6. Incubate for 1 h at room temperature.
    7. PCR DNA cleanup from New England Biolabs to clean the DNA in ligation mix from any impurities.
      1. Add 70 uL of binding cleanup buffer to the eppendorf tube.
      2. Transfer the 80 uL solution into a column and insert the column in a collection tube.
      3. Centrifuge the tube for 1 min. Discard flow-through and reinsert column into collection tube.
      4. Add 200 uL of wash buffer in column and centrifuge for 1 min. Discard is optional.
      5. Repeat the previous step. Discard flow-through and collection tube.
      6. Insert column into an eppendorf tube and add 6 uL of elution buffer. Incubate for 1 min before centrifuging. Discard column.
  3. Amplification mix:
    1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
    2. Add 2 uL of diluted FIP and 2 uL of diluted BIP.
    3. Add 6 uL of water.
    4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 h at 65°C.

LAMP - Antibodies

His-tag antibodies were used to capture tau in tears. High-binding 96-well plates were coated with antibodies, solution containing tau was added, aptamer was added and then LAMP was performed. To get reliable results triplicates of antibody + tau were prepared, as well as triplicates of just tau coating wells. Finally, blanks were also used.

Preparations

  • PBS: 1 PBS tablet + 1 L deionized water.
  • PBS-T: mix 300 mL pBS with 150 uL of Tween.
  • 2% BSA: mix 0.024 g of BSA powder in 12 mL of PBS.

Protocol

  1. Plate coating:
    1. Mix 120 uL of antibody with 2280 uL of buffer.
    2. Added 100 uL of the mix to 6 wells (triplicates + blanks) in a 96-well plate.
    3. Serial dilution of dialysed tau was done to get tau concentration of 1/16 * 3.2 g/L = 0.2 g/L.
    4. Add 100 uL of diluted tau to 3 wells.
    5. Cover the plate with plastic and incubate overnight at 4°C.
  2. Tau addition:
    1. Wash all wells 4 times with 100 uL PBS-T.
    2. Add 100 uL of 2% BSA per well.
    3. Cover with plastic and incubate for 1h at room temp.
    4. Repeat the serial dilution of tau and add 100 uL of 0.2 g/L tau to 3 of the wells coated with antibody.
    5. Cover with plastic and store in the fridge overnight.
  3. Aptamer-linker addition:
    1. Wash 4x with 100 uL of PBS-T per well.
    2. Dilute 12.5 uL of 100 uM aptamer in 4987.5 uL of BSA.
    3. Add 100 uL of diluted aptamer to each well.
    4. Cover plate with plastic and incubate for 2h at room temp.
    5. Wash 4x with PBS-T.
  4. Ligation mix:
    1. Mix in an eppendorf tube 10 uL of 10x T4 DNA ligase buffer, 17 uL of phosphorylated linear padlock probe, 10 uL of T4 DNA ligase, 13 uL of sterile water.
    2. Add 10 uL of mix to each well.
    3. Incubate for 1 hour at room temperature.
    4. No PCR DNA cleanup is performed.
  5. Amplification mix:
    1. Mix in an eppendorf tube 200 uL of 2x WarmStart colorimetric LAMP master mix, 20 uL of diluted FIB, 20 uL of diluted BIP, 60 uL of sterile water.
    2. Add 30 uL of the mix to each well. Incubate for 1 hour at 65°C.

Agarose gel

To validate the purity and amplification of the plasmid an agarose gel was used. Agarose gel is used to separate macromolecules, such as DNA, based on size and charge. This was also done to verify the binding of aptamer to protein.

Preparations

  • Add 50 mL of 10x TAE and 450 mL deionized water into a 500 mL flask.
  • Add 1g of agarose powder into 100 mL of 1X TAE to make a 1% agarose gel for plasmid verification. For LAMP validation, 1.5 g agarose powder was added to 100 mL 1 x TAE to make a 1.5% agarose gel. Heat the solution in the microwave for 2 minutes until it is completely transparent.
  • Before pouring the agar solution into the gel mould, let it cool down a little. Insert a comb and let the gel set for 20-30 min.

Protocol

  1. Plasmid analysis
    1. Centrifuged the purified plasmid for a couple of seconds.
    2. Add 11uL of water and 4uL of 6x loading dye into a tube containing 5uL of the plasmid.
    3. Load the samples on the agarose gel and as a reference 5uL of a DNA ladder.
    4. Insert the gel in an electrophoresis cassette and add 1X TAE buffer until it covers the gel. Run the gel at 100 V for one hour.
    5. Stain the gel in ethidium bromide for 5-10 min.
    6. Rinse the gel in deionized water.
    7. Visualise the DNA bands by a UV machine at 302 nm.
    8. Take a picture.
  2. LAMP verification
    1. Centrifuged the aptamer for a couple of seconds.
    2. Add 16uL of aptamer solution water and 4uL of loading dye into a tube.
    3. Load 5 uL of ladder and 20 uL of samples into the agarose gel for plasmid validation.
    4. Insert the gel in an electrophoresis cassette and add 1X TAE buffer until it covers the gel. Run the gel at 100 V for one hour.
    5. Stain the gel in ethidium bromide for 5-10 min.
    6. Rinse the gel in (a container of) deionized water.
    7. Visualise the aptamer bands by a UV machine at 302 nm.
    8. Take a picture.

MST

MST is a method to check the affinity between a fluorescent-labelled protein and a non-fluorescent ligand. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. Interactions are detected by changes in molecular diffusion or fluorescence intensity. In this case, the target whose change in fluorescence is observed is the tau protein, which was labelled using Red-tris-NTA dye (interacting with the His-tagged tau protein), and the non-fluorescent ligand is the aptamer with linker.

Preparations

  • PBS: 1 PBS tablet + 1 L deionized water.
  • PBS-T: mix 300 mL PBS with 150 uL of Tween.
  • 50 nM Dye: 2 uL of 5 uM Red-Tris NTA mixed with 198 uL of PBS-T.
  • 100 nM Dye: 2 uL of 5 uM Red-Tris NTA mixed with 98 uL of PBS-T.
  • His-tagged protein: make a 4 uM tau solution in PBS-T, volume 30 uL by mixing 2,6 uL of 45,3 uM tau in 27,4 uL PBS-T.
  • His-tagged protein: make a 4 uM tau solution in PBS-T, volume 100 uL by mixing 8,8 uL of tau in 91,2 uL PBS-T.

Protocol

  1. Affinity between dye and tau:
    1. Prepare 16 PCR tubes. Add 20 uL of 4uM tau solution in tube 1. In tubes 2-16, 10 uL of PBS-T.
    2. Transfer 10 uL of tau solution from tube 1 to tube 2 and mix by pipetting up and down. Continue with the dilution series by repeating the process for all tubes. Discard 10 uL from tube 16 to have the same volume in all.
    3. Add 10 uL of 50 nM dye to all tubes. Incubate for 30 min at room temp.
    4. Load capillaries with samples from each tube. Simply put the capillary in the tube until the solution has occupied its length.
    5. Run MST program and check results.
  2. Protein labeling:
    1. Mix 90 uL of 4 uM tau with 90 uL of 100 nM dye in an eppendorf tube.
    2. Incubate for 30 min at room temperature.
    3. Centrifuge at 13400 g for 10 min.
  3. Binding assay:
    1. Prepare a 2 uM aptamer solution by mixing 0,5 uL of 100 uM aptamer with 24,5 uL PBS-T.
    2. Prepare 16 PCR tubes. Add 20 uL of aptamer solution in tube 1. In tubes 2-16, 10 uL of PBS-T.
    3. Perform serial dilution, as done in step 1.2.
    4. Add 10 uL of RED-tris-NTA-labeled tau protein to each tube and mix by pipetting up and down.
    5. Load capillaries and run MST program.

SDS-PAGE

SDS-PAGE was used to verify tau protein expression and purity. The SDS method requires multiple components like gels, buffers and the gel electrophoresis apparatus.

Preparations

  • SDS buffer: Prepare 1 L of Tris glycine SDS-buffer by diluting 10X stock solution to 1X with deionized water. Final concentrations are as follows: 25 mM Tris, 192 mM glycine and 0.1% SDS. Adjust pH to 8.3.
  • Add two gels in the cassette holder. Test for leaks by adding a buffer in between the gels first. Repeat if using more than two. If only one gel is added, one blank needs to be added as well.
  • Add the cassette holder into the SDS apparatus, and pour the SDS buffer all the way in between the gels and up to the demarcation line.
  • Destaining solution: Prepare 1L of solution by adding 500 mL water, 400 mL methanol and 100 mL acetic acid into a plastic bottle.

Protocol

  1. Run gel:
    1. Prepare samples in a fume hood by mixing 5uL of 4X SDS-buffer(containing gel-loading dye bromophenol blue) with 1uL of BME and 14 uL of sample solution.
    2. Heat samples on a heating block at 95°C for 5 min.
    3. Load 10 uL of samples and 3uL of ladder into the wells of the gel.
    4. Run the gels at 200 V until migration of the samples into the gel is observed, then increase the voltage to 300 V for 25 min or until the bands reach the bottom of the gel.
  2. Staining the gel:
    1. Remove the gel from the holder and place it into a smaller container. Add deionized water carefully to the container. Carefully pour off the deionized water.
    2. Transfer to a fume hood.
    3. Add just enough Coomassie Brilliant Blue R-250 Dye staining solution to cover the gel and put the container on a shaker. Leave the container on the shaker. The shaker should be set to gently shake the samples.
    4. Destain the gel by pouring off the staining solution and by adding destaining liquid.
    5. Add tissue to the side of the container to soak up more stain.
    6. Change both tissue and destaining solution periodically until the gel is transparent and the protein bands show clear contrast to background.

    3. *Note that we also analyzed our gel using a fluorescence imager with a 480 nm excitation filter. This was possible because we expressed a tau-GFP fusion protein.

Synthetic tears

As using human tears could be limited, synthetic tears were made that closely resemble human tears. Synthetic tears with and without tau were made. Another advantage is that synthetic tears make it easier to quantify the results and troubleshoot experiments.

Preparations

  • NaCl stock solution (1:10) 1,25 mol/L.
    • Add 1.825 g NaCl to 25 mL Water.
    • Mix and label the samples.
  • CaCl2 stock solution (1:10) 14 mmol/L.
    • Add 0.075g CaCl2 to 50 mL water.
    • Mix and label the sample.
  • MgCl2 stock (1:10) 20 mmol/L.
    • Add 0.0935 g MgCl2 to 50 mL water.
    • Mix and label the sample.
  • Glucose stock solution (1:100) 20 mmol.
    • Add 0.9g glucose to 25 mL water.
    • Mix and label the sample.
  • KCl stock solution (1:10) 0,16 mol/L.
    • Add 0,12g KCl to 50 mL water.
    • Mix and label the sample.
  • All stock solutions can be stored in room temperature.

Protocol

  1. Make the synthetic tears
    1. Add 5 mL NaCl stock solution.
    2. Add 5 mL CaCl2 stock solution.
    3. Add 5 mL MgCl2 stock solution.
    4. Add 5 mL KCl stock solution.
    5. Add 500 uL glucose stock solution.
    6. Add water up to 50 mL.
    7. Divide into 2 vials.
    8. Add 0.25 g of luophilized bovine serum albumin and 0.075 g of lyophilized hen egg white lysozyme to solution.
    9. Store unused stock solution at 4°C.
  2. Add tau to the tears.
    1. Take 5 mL of finished tears to test tubes.
    2. Add tau from the stock solution to concentrations of 10 ng/mL, 5 ng/mL, 1 ng/mL, and leave one as is, and add deionized water.
    3. Store the solutions at 4°C.

Tau production

Before we can use our tau protein it needs to be synthesised and purified first. The production of tau is divided into three stages namely plasmid transformation, tau expression and lastly purification. At some of the intermediate steps, samples were taken and frozen for troubleshooting if something went wrong in later steps. Two strains of E.coli competent cells were used. One for the the purpose of the production of tau (NEB BL21/DE3) and another strain that will later be used for plasmid preparation (NEB DH5-α).

Preparations

  • LB medium: Make 2 batches of 1L LB medium by adding 20 g of LB-mix into a 1 L flask and top it up with deionized water until the 1 L mark is reached.
  • LB agar: Make 1L of LB-agar by adding 15 g of agar powder to a 1 L flask and top it up with LB medium until the 1 L mark is reached.
  • Kanamycin stock solution: Add 0,3 g of Kanamycin into a 10 mL volumetric flask and fill up to the 10 mL mark so that the final concentration is 30 mg/mL. The 10mL stock solution was aliquoted into 1,5 mL eppendorf tubes.
  • Sterile Water: Pour 250mL deionized water into a 500 mL flask and autoclave it.
  • 1 mM of IPTG.
  • Elution buffer: Make 0,5 L of elution buffer by adding 3,02 g of 50 mM Tris, 17,02 g of 20 mM Imidazole and 8,77 g of 300 mM NaCl and then filling up the flask until the 500 mL mark is reached. The elution buffer should have a pH of 7. If the solution doesn’t have this specific pH it needs to be adjusted.
  • Equilibration buffer: Make 1 L of equilibration buffer by adding 6,03 g of 50 mM Tris, 1,36 g of 20 mM Imidazole and 17,53 g of 300 mM NaCl and then filling up the flask until the 1 L mark is reached. The elution buffer should have a pH of 8. If the solution doesn’t have this specific pH it needs to be adjusted.

Protocol

  1. Agar plates:
    1. Melt LB agar in the microwave until it is completely liquid.
    2. Pour LB agar into sterile petri dishes until the bottom is covered.
    3. Put the lids on halfway so that half of the petri dish is open, to allow it to set.
  2. Plasmid transformation:
    1. Check the concentration of the tau plasmid by using a Nanodrop. Dilute the plasmid using sterile water to the wanted concentration if the plasmid is not already in the wanted concentration.
    2. Thaw two tubes of NEB DH5-ɑ competent E.coli cells and NEB BL21/DE3 competent E.coli cells on ice for 10 min. Pipette 1 uL of the plasmid mixture into the two tubes.
    3. Leave the tubes on ice for 30 min and then heat shock at 42°C for 10 seconds.
    4. Leave the tubes on ice for 5 min.
    5. Pipette 250 uL of room temperature SOC into each of the tubes.
    6. Incubate the tubes at 37°C for 1 hour with vigorous shaking at 250 rpm.
    7. Spread both of the strains on 6 agar plates each and put in 37°C incubator overnight.
  3. Plasmid culturing and Tau expression:
    1. Prepare a preculture: add 20 mL of LB medium, 20 uL of kanamycin and 1 colony of BL21/DE3 into a 50 mL Falcon tube. Repeat this step for the DH5-ɑ strain. The remaining colonies on the agar plate can be sealed with Parafilm and stored at 4°C until further use.
    2. Incubate the tubes at 37°C overnight with vigorous shaking at 240 rpm. In the meantime, prepare and autoclave 1 L of LB-medium.
    3. Add 5 mL of glycerol into the falcon tube containing the DH5-ɑ cells and flash freeze it in liquid nitrogen for 2-4 seconds. Store the sample in -80°C.
    4. Put 1 mL kanamycin and 10 mL of preculture in the autoclaved LB medium.
    5. Take 1 mL of the culture and measure O.D. in O.D.-meter. This sample serves as blank.
    6. Divide the BL21/DE3 culture equally into two falcon tubes.
    7. Incubate for 90 min at 37°C at 90 rpm. Measure O.D. of 1 mL from each tube and compare with blank.
    8. Add 0.5 mL of 1 mM IPTG into each falcon tube.
    9. Incubate the tubes at 37°C for 4h or 16°C overnight at 90 rpm.
  4. Tau Purification:
    1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
    2. Add 2 uL of diluted FIB and 2 uL of diluted BIP.
    3. Add 6 uL of water.
    4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 hour at 65°C.
  5. Protein dialysis:
    1. Prepare a 14 kDA membrane by cutting approximately 10 cm of the membrane and place it in a beaker filled with deionized water.
    2. Transfer the membrane into a plastic container and add two clamps on one side of the membrane.
    3. Open the membrane on the open side carefully and pippet the tau elution into the membrane.
    4. Add 2 clips to the open end of the membrane.
    5. Add the now filled membrane into a beaker containing dialysis buffer.
    6. Place the beaker at 4°C on a magnetic stirrer for 1 hour.
    7. Remove the dialysis buffer and replace it with TAE dialysis buffer containing Sodium Azide.
    8. Keep the beaker on a magnetic stirrer overnight.
    9. Remove the clips on one side of the membrane and pipette out the contents into a 15 mL Falcon tube.

Plasmid preparation

In order to check the purity and if the plasmid that is being used has the wanted construct plasmid preparation was done. The NEB DH5-α strain of E.coli competent cells were used for plasmid preparation.

Preparations

  • Plasmid preparations should already have been done for the NEB DH5-α strain.
  • A plasmid cleanup kit such as the Monarch® Plasmid Miniprep Kit from New England BioLabs is needed.
  • LB medium: Make 1 L LB medium by adding 20 g of LB-mix into a 1L flask and top it up with deionized water until the 1 L mark is reached.
  • Kanamycin: Thaw an already made aliquot of 30 mg/mL kanamycin until it is completely liquid.

Protocol

  1. Bacterial growth
    1. Add 10 mL of LB-medium and 10 uL of Kanamycin as well as 2-3 colonies of DH5-α into a 50 mL Falcon Tube.
    2. Incubate the tube at 240 rpm at 37°C for 4-5 hours.
  2. Plasmid preparation
    1. Pour 1 mL of the culture into a 1,5 mL eppendorf tube.
    2. Centrifuge the tube for 1 min and discard the supernatant.
    3. Use a plasmid cleanup kit such as the Monarch® Plasmid Miniprep Kit to isolate the plasmid.
    4. The plasmid can now be sent for sequencing.
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