IGEM:Harvard/2006/Gel staining

From OpenWetWare
Jump to navigationJump to search

EtBr staining of mini PA gels

Incubate in 10μL EtBr + 100 mL Tris-glycine buffer for 30 min.

Coomassie staining of mini PA gels

Protocol from Pierce using GelCode Blue:

  • 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.

Silver staining

Protocol using Bio-Rad Silver Stain Plus Kit and Bio-Rad's protocol:

  • fix gels for 20 to 50 minutes in fixative enhancer solution
    • fixative enhancer solution: 200 mL MeOH, 40 mL Bio-Rad fixative enhancer concentrate, 40 mL glacial acetic acid, 120 mL water
  • rinse in water for 10 minutes, replace water, rinse again for 10 minutes
    • the gel has a tendency to curl up during this step, and so you may have to manually uncurl it, or turn it over in the water Matthewmeisel
  • 5 minutes before use, place 35 mL water in a flask, and add, in order, with stirring: 5 mL Bio-Rad silver complex solution, 5 mL Bio-Rad reduction moderator solution, and 5 mL Bio-Rad image development reagent
  • immediately before use, add 50 mL of room temperature Bio-Rad development accelerator reagent (ordinarily stored at 4[[:Category:{{{1}}}|{{{1}}}]])
  • stain gels for desired amount of time in this mixture
  • stop staining in 5% acetic acid solution, incubate for 15 min.
  • rinse with water for 5 min.

SYBR gold staining

Protocol from Invitrogen:

  • staining solution: 10 μL of 10,000x Invitrogen SYBR Gold in 100 mL TBE buffer, stored in dark plastic container at 4[[:Category:{{{1}}}|{{{1}}}]]
  • incubate gel in staining solution for 10 to 40 min.
  • staining should be carried out in dark (wrap aluminum foil around container)
  • staining solution may be reused if desired for two to three more gels