IGEM:Harvard/2006/DNA nanostructures/Protocols

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Aptamer-decorated Nanostructure Binding Assay Potential protocol for a 2 μL incubation reaction (revised with Dr. Shih's suggestions)

• In a 0.2 mL PCR tube, mix:
  • 0.5 μL of 4x (not 5x) Bock's selection buffer
  • 1.0 μL of 2 μM aptamers (final concentration: 1.0 μM = 2 pmol)
  • 0.5 μL of 2 μM thrombin (final concentration: 0.5 μM = 1 pmol)
• OR in a 0.2 mL PCR tube, mix:
  • 0.5 μL of 4x (not 5x) Bock's selection buffer
  • 0.5 μL of 2 μM aptamers (final concentration: 0.5 μM = 1 pmol)
  • 1.0 μL of 2 μM thrombin (final concentration: 1.0 μM = 2 pmol)
• Alternative mix: Liu uses 10 pmol of DNA (1 μL of 10 μM) and varies thrombin amount from 2 pmol (1 μL of 0.2x thrombin working stock) to 100 pmol (1 μL of 10x thrombin working stock)
• Incubate at room temperature for 30 min.
• Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4[[:Category:{{{1}}}|{{{1}}}]]
  • Run at 130 V for approximately 90 min.
  • Liu runs at 25 mA for 48 h.

Staining Native gels - protocol from Pierce
Procedure for Staining Gels

• Wash
  • Native PAGE: Rinse gel with ultrapure water for 5 minutes.
  • Note: Gels electrophoresed with MOPS or MES running buffers must be prefixed with a 50% methanol and 7% acetic acid solution for 15 minutes and then washed with ultrapure water to remove fixing solution. GelCode ® Blue Stain penetrates prefixed gels better than non-fixed gels and, therefore, standard SDS-PAGE gels may also be prefixed with good results.

• Stain Note: Mix the GelCode ® Blue Stain Reagent solution immediately before use by gently inverting or tipping and swirling the bottle several times. Such mixing is especially important when using Product No. 24592 with a dispenser pump. Do not shake bottle to mix the solution.

• • Add 20 ml of GelCode ® Blue Stain Reagent for an 8 x 10 cm mini gel. Additional reagent may be required if a large tray is being used. Gently shake tray and periodically monitor protein band development. Stain intensity reaches a maximum within approximately 1 hour. Gels may be stained overnight without increasing background.

• • Note: PhastGel ® Gels may require increased staining times (2 hours to overnight) for optimal band development.

• Destain (Water Wash EnhancementTM Step) Replace Stain Reagent with ultrapure water. Several water changes for a 1-2 hour period may be necessary for optimal results. This step enhances stain sensitivity, as weak protein bands continue to develop.