Team:Freiburg/Protocols/APTES PDITC

""

<< Back

APTES + PDITC surface

Protocol for derivatisation of glass slide with APTES and coupling of amino groups
According to protocol from Christin (AG Roth)

material: chemicals, used kits, …
duration: … min

Slide preparation

  1. Name the slide
  2. Wash with: acetone, isopropanol, aqua dest.
  3. Dry with wafergun
  4. Activate in plasma generator: gas flow 80 L/h, time 2 min

APTES

  1. Prepare APTES-solution: for 5 slides:
    • 18.8 mL acetone
    • 1.0 mL dd aqua
    • 0.2 mL APTES (always close with Parafilm)
  2. Incubate slides for 30 min in slideholder filled with APTES-solution
  3. Wash 2 x 5 min in slideholder with acetone
  4. Dry with wafergun
  5. Put slides in new slideholder and bake them in oven at 110 °C for 45 min

PDITC

  1. Prepare PDITC-solution: for 5 slides:
    • pyridin 2.0 mL
    • DMF 18.0 mL
    • PDITC 40 mg
  2. Cool down slides in slidebox with wafergun (go close with gun)
  3. Incubate slides in slideholder for 2 h filled with PDITC-solution at RT
  4. Discard PDITC-solution
  5. Fill with ethanol and discard ethanol
  6. Wash 2 x 5 min in slideholder with ethanol
  7. Wash for 5min with acetone in slideholder
  8. Discard acetone
  9. Dry with wafergun
  10. Dry for 15 min in desiccator (Exsiccator)

Spotting

  1. Prepare 2 µM bBSA-solution (diluted from stock solution)
  2. Put humid tissues inside petri-dish
  3. for DNA spotting control use Spotting control Cy5 protocol
  4. Spot 0.5 µL of DNA-control-solution (2 spots) and bBSA-solution (3 spots).

Use spotting templates!

  1. Close petri-dish with Parafilm
  2. Incubate overnight

Blocking

  1. Prepare spots for blocking:
    • let spots become dry on air
    • wash immediately with aqua dest. and dry with wafergun
  2. Prepare ethanolamine-solution (10 %, 20 mL):
    • 2 mL ethanolamine
    • 18 mL aqua dest.
  3. Incubate slides for 30 min in slideholder filled with ethanolamine-solution
  4. Fill with aqua dest. and discard aqua dest.
  5. Wash 2 x 5 min with aqua dest.
  6. Dry with wafergun
  7. Pipette 80 µL of BSA-solution in PBS-buffer (10 mg/mL) per slide-sandwich
  8. Incubate for 30 - 60 min
  9. Wash with aqua dest.
  10. Dry with wafergun

Binding of Strep-Cy5

  1. Prepare Streptavidin-Cy5-solution (0.5 µg/mL)
    • 180 µL BSA in PBS-buffer (10 mg/mL)
    • 20 µL Strep-Cy5-stock solution (5 µg/mL)
  2. Pipette 80 µL of Strep-Cy5-solution per slide-sandwich
  3. Incubate for 45 min at RT (Put a tissue over petri dish)
  4. Wash with aqua dest. by letting glide slidese into vase filled with aqua dest.
  5. Dry with wafergun


  1. Fluorescence detection