DNA Reduction and Immobilization

From LPDwiki

Necessary Materials

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

2. Aliquoted probe DNA (C = 100 μM, V = 35 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

7. 1 mM MCH (6-Mercapto-1-hexanol)

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

Procedure

DNA Probe Thiol Reduction and Quantification

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. Keep 20 μl aside for control absorbance measurement of the unreduced probe.

4. Add 4 μl of 0.5 M TCEP to 100 μl of unreduced DNA probe solution (~25 μM). Mix and vortex for 5-10 seconds.

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

  • Cover with Al foil.

6. Filter the mixture using zeba spin column. Centrifuge at 4000 rpm for 2 minutes.

  • Note, zeba spin columns need to be centrifuged at 4000 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.

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

  • Expected concentration after reduction ~ 30 μM.

DNA Probe Immobilization

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.

3. Plasma treat electrodes for 1 minute prior to probe functionalization.

4. Incubate electrode with 5 μM DNA probe immobilization solution (100 μl of 25 mM PB + 400 μl of 75 mM MgCl2 + 100 μl of 30 μM reduced probe DNA) for 15 hours at room temperature.

  • Volume of different components of 5 μM DNA probe immobilization solution was determined using the following specifications:
    • Vfinal = 600 μl
    • C(probe DNA)final = 5 μM
    • C(MgCl2)final = 50 mM
    • Rest is 25 mM PB
  • Cover and parafilm the petri-dish containing the sample with immobilization solution.

5. Rinse 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.

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