Yu:High-resolution gels

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Contents

Making the Gel

  1. Treat the notched glass plate with Glass-Free
    1. To apply Glass-Free clean glass plate with Detergent, dH2O, Methanol.
    2. You can either soak the plate in Glass-Free for 5-10 min or buff the glass plate with Glass-Free. Follow this with a quick cleaning with methanol.
  2. Clean the other glass plate with Ethanol and let both plates dry completely.
  3. Clean the comb and spacers with ethanol as well and let dry.
  4. Make a batch of ~2% agarose with dH2O (this can be saved and reused until gone)
  5. Assemble plates with spacers. The whole set-up can be held together with black binder clips.
  6. Once the agarose has cooled some, use a plastic Pasteur pipet to apply the agarose to the outer edges of the gel plates.
 Note: This acts to seal the plates and spacers so that the Polyacrylamide will not leak.  
  1. Mix the appropriate ratios of SequaGel monomer solution and SequaGel complete buffer in a 50 mL falcon tube; mix well. Add the 10% APS (from SDS-PAGE supplies, there is an aliquot in the 4˚C fridge).
 Note: Save the last pipet you use for pouring the gel.  Our largest gel plates hold ~15-18mL so make 20mL of solution
  1. Vortex Briefly
  2. Pour the gel so that the liquid is almost to the top of the lowest plate and then insert the comb ensuring that there are no air bubbles in the wells.
 Note: It is best to set the gel plates at a 45˚ angle when pouring.
  1. Save the left over gel solution to monitor polymerization (~1-2 hr.)

RNA sample preparation

  1. You can use anywhere from 10-30 µg of RNA
  2. Mix with at least an equal volume of Loading Dye
  3. Incubate samples at the prescribed temperature and for the indicated time.
 Note: Be sure to treat the Ladder the same as the samples (load 0.5-1 µg), check manufacturer’s insert for the specific dye for incubation conditions

Running the Gel

  1. Pre-run the gen for 30 min. at 70V
 Note: Use 1x TBE as your running buffer
  1. Rinse the excess Urea, from the wells
  2. Load the samples as gently as possible
  3. Run the gel at 70V for ~8 hr. or run at 200V for ~1.5 hrs.
 Note: If you run the gel at 200V, be sure to run it in the cold room

Staining the Gel

  1. If the loading Dye doesn’t already have EtBr in it, stain the gel in 500mL of H2O + EtBr for 10 min.
  2. Rinse in H2O and photograph, be sure to include a ruler in the photograph

Semi-Dry Elctroblotting

  1. Cut out 8 sheets of whatman paper to fit the gel, cut membrane out to the same size or just a little bit smaller.
  2. Soak the whatman paper, membrane and gel in 1x TBE for 30 min.
 Note: Be sure that the Gel and membrane do not touch while soaking, you do not want an random transfer of nucleic acid to the membrane!!!
  1. Set-up the blotting apparatus (BioRad: Transblot SD)
 From the bottom: 4 pieces of Whatman paper, membrane, gel, 4 pieces of whatman paper.  Be sure to role out the bubbles in each layer.
  1. Set the current at 1.33 mA/cm2
  2. Check gel with Gel-doc after blotting to ensure a good transfer has taken place.
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