DNA Reduction and Immobilization

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Necessary Materials

1. np-Au/planar Au chips (22 mm X 22 mm) → working electrode

2. Aliquoted probe DNA (C = 100 μM, V = 30 μl)

3. 0.5 M TCEP (tris(2-carboxyethyl)phosphine)

4. Zeba Spin Desalting Columns

  • Product Number: 89882

5. 25 mM Phosphate Buffer (PB)

6. 75 mM MgCl2: Always prepare fresh solution!

7. 1 mM MCH (6-Mercapto-1-hexanol): Always prepare fresh solution!

8. Platinum wire → counter/auxillary electrode

  • Product Number: 45093
  • 0.25 mm (0.010 in) dia, 99.9% (metals basis) length: 50 cm

9. Reference Electrode

  • Product Number: ET073-1
  • Refillable miniature Ag/AgCl reference electrode

10. PCR centrifuge tube, RNase-free

Procedure

All solutions should reach room temperature before being used.

DNA Probe Thiol Reduction and Quantification, V = 120 μl, C = 20 μM

1. Take out probe aliquot from -20 °C and let it thaw and equilibrate at room temperature for 20 minutes.

2. Add 30 μl of the DNA stock solution to 90 μl of 25 mM PB to prepare 25 μM dilution. Vortex gently (5 seconds) and spin down (4 seconds).

3. Add 4 μl of 0.5 M TCEP to 120 μl of unreduced DNA probe solution (~25 μM). Mix and vortex for 5-10 seconds.

4. Leave reaction mixture at room temperature for 2 hours.

  • Cover with Al foil.

5. Filter the mixture using zeba spin column. Centrifuge at 4,000 rpm for 2 minutes.

  • Note, zeba spin columns need to be centrifuged at 4,000 rpm for 2 minutes to get rid of desalting solution, before DNA solution is added.
  • Make sure to label formed slanted side before adding DNA solution. Labelled slanted side should face outside in the microcentrifuge during DNA centrifugation.
  • Don't forget to balance the microcentrifuge.

6. Measure absorbance of reduced DNA probe using Biotek UV-Vis.

  • Expected concentration after reduction ~ 20 μM.

7. Label the RNase-free tube: 20 μM TCEP reduced probe DNA, V = 120 μl (Initial, Date) IMPORTANT: TCEP reduced DNA probe can be stored in the freezer for 7 days maximum. Make sure not to add any salt to the stock solution - add salt only to the DNA probe immobilization solution that is to be used on the given day.

DNA Probe Immobilization: e.g. V = 600 μl, C = 1 μM

1. Cut np-Au/planar Au chips (22 mm X 22 mm) into four squares. Use one square per experiment.

2. Prepare piranha solution (1 : 4; hydrogen peroxide: sulphuric acid) and leave it to cool for 10 minutes. Clean the electrodes by immersing for 20 seconds each. Rinse with DI water and store in DI water until DNA probe functionalization.

  • Alternatively, watch the chips with ethanol and DI/MilliQ water.

3. Plasma treat electrodes for at least 1 minute prior to probe functionalization (use plastic petri-dish cover for plasma cleanin).

4. Incubate electrode with 1 μM DNA probe immobilization solution (170 μl of 25 mM PB + 400 μl of 75 mM MgCl2 + 30 μl of 20 μM reduced probe DNA) for 15 hours at room temperature.

  • Volume of different components of 1 μM DNA probe immobilization solution was determined using the following specifications:
    • Vfinal = 600 μl
    • C(probe DNA)final = 1 μM
    • C(MgCl2)final = 50 mM
    • Rest is 25 mM PB
  • Cover and parafilm the petri-dish containing the sample with immobilization solution. If possible, there should be a separate well with PB included in the para-filmed setup to prevent probe solution evaporation overnight.

IMPORTANT: Always prepare fresh 75 mM MgCl2!!! 75 mM Magnesium Chloride Solution Preparation

5. Rinse the samples with 25 mM PB to remove non-specifically bound probe molecules. Transfer the electrode to a petri dish containing 5 ml of 25 mM PB.

6. Incubate the electrode with back-fill agent, 1 mM mercaptohexanol (MCH) prepared in 25 mM PB for 2 hours to passivate the surface that has not been covered by probe DNA. IMPORTANT: Prepare fresh 1 mM MCH every time!!! 1 mM MCH Solution Preparation

7. Rinse electrodes thoroughly with 25 mM PB to remove non-specifically bound DNA.

8. Dry the functionalized electrode with nitrogen, holding the electrode parallel to the gas stream before assembling in the electrochemical cell for DNA hybridization experiment.