Team:Freiburg/Protocols/Glassslide

• Home
• Project
• Team
• Results
• Human Practice
• Notebook

• Introduction
• Overview
• Diagnostics today
• The DiaCHIP
  • DNA Engineering
  • Cell-Free Expression
  • Protein Purification
  • Surface Chemistry
  • Optical Detection
• Outlook

• Members
• Acknowledgements
• Partners

• The DiaCHIP
  • Main Results
  • Diagnostics
  • Our Device
  • DNA Array
  • Cell-Free Expression
  • Binding on Surface
  • Modeling
• BioBricks
• Collaborations
• Interlab Study
• Achievements

• Science Fair
• Theater Project
• Public Opinion
• College Talk
• Ethics

• Labjournals
• Techniques
  • Gibson
  • E.coli Strains
• Protocols
• Materials
• Safety
• Wiki-Tricks
• Glossary

• Home
• Project
• Results
• Team
• Practice
• Notebook

Menu

<< Back

Glass slide preparation

complete protocol for functionalization of glass slide with APTES/PDITC and Ni-NTA

duration: 2 days

---

Slide preparation

• Name the slide
• Wash with: acetone, isopropanol, aqua dest.
• Dry with wafergun
• Activate in plasma generator: gas flow 80 L/h, time 5 min
  

APTES

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

PDITC

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

Ni-NTA

• prepare 80 µL 457 mM AB-NTA, 1 M NaHCO₃ solution per slide sandwich
  • use 0.06 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.042 g NaHCO₃ in 500 µL aqua dest
  • use 0.096 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.067 g NaHCO₃ in 800 µL aqua dest
  • use 0.108 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.076 g NaHCO₃ in 900 µL aqua dest
• put 80 µL on one slide and put the other one upon → Make sure the upper sides were laid together
• incubate o/n
• prepare blocking solutions:
  • blocking solution for GOPTS: 10mg/ml BSA + 0.5% ethanolamine in PBS
  • blocking solution for APTES/PDITC:5mg/ml BSA + 5% ethanolamine in PBS
• transfer slides into blocking solution for 30 min-60 min
• 2x wash slides for 10 min in PBS (shake it)
• prepare 20 mL 1 % (w/v) NiSO₄ solution per 5 slides (= 0.2 g/20 mL)
• incubate slides in slide holder with nickel sulfate for 1 h
• 2x wash slides for 10 min in PBS (shake it)
• prepare 1/10 PBS-dilution in aqua dest and check for pH (should be 7-8)
• wash slides in diluted PBS-solution
• Dry slides with wafer gun

Spotting and washing

• spot using 11-spot mask
• (use 3 µl of bBSA (200 µg/ml in 1mg/ml BSA) as control spot)
• incubate o/n @ 4°C
• remove protein solution with pipette
• incubate twice 5 min with 10 mg/mL BSA solution in the wells
• (flush slide with PBS)transfer slide into PBS solution and remove mask
• wash 10 min in PBS
• wash 10 min in PBS (with 20 mM Imidazol, 27.2 mg in 20 mL PBS)
• wash 10 min in 1/10 diluted PBS
• dry with wafer gun
• measure fluorescence
• incubate for 45 min in Strep-Cy5 solution (5 µg/ml)+ 5-10mg/ml BSA
• 2x wash 10 min in PBS (shake it)
• wash 10 min in 1/10 diluted PBS
• dry with wafer gun
• measure Cy5-fluorescence in microarray scanner

protocols, surchem