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12.04 Loading dyes

Loading dyes are mixed this samples to pull them down into a well in a gel and to visually track migration during electrophoresis.

When using fluorescently-labelled (Cy3, Cy5) samples, make sure the tracking dyes do not co-migrate with your sample, otherwise you may be unable to tell if a resulting band originates from your sample or the dye.

For native PAGE, do not use dyes that contain SDS as they may separate double-stranded nucleic acids.

Common dyes (separate)

Bromophenol blue

  • 3′,3″,5′,5″-tetrabromophenolsulfonphthalein (BPB)
  • Below pH 3.5: yellow
  • Above pH 4.6: blue
  • Slightly negative
  • 1% agarose gel in TAE or TBE: co-migrates with 300 bp DNA
  • 2% agarose gel in TAE or TBE: co-migrates with 150 bp DNA

Xylene cyanol

  • 1% agarose gel: co-migrates with 4-5k bp DNA
  • 6% polyacrylamide gel: co-migrates with 140 bp DNA
  • 20% denaturating (7 M urea) PAGE: 25 bp DNA

Orange G

  • Orange in pH < 8
  • Red in pH > 9

Common dye mixes

TriTrack

Source

  • Contents:
    • 10 mM Tris-HCl (pH 7.6)
    • 0.03 % bromophenol blue
    • 0.03 % xylene cyanol FF
    • 0.15 % orange G
    • 60 % glycerol
    • 60 mM EDTA.
  • Uses:
    • Agarose gel
    • Native PAGE

Gel Loading Buffer II

Source

  • 1-2X solution
  • Contents:
    • 95% Formamide
    • 18 mM EDTA
    • 0.025% each of SDS, Xylene Cyanol, and Bromophenol Blue
  • Uses:
    • Denaturing PAGE
    • Non-denaturing agarose gel
  • Appearance: Dark blue

Protocol:

  1. Mix sample with an equal volume of Gel Loading Buffer II. Vortex briefly.
  2. Centrifuge briefly to bring contents of tubes to the bottom.
  3. For denaturing PAGE: Heat to 95°C for 5 min to denature any secondary structure.
  4. Load directly (while still hot) on the gel.

RNA Loading Dye

Source

  • 2X
  • Contents:
    • 47.5% Formamide
    • 0.01% SDS
    • 0.01% bromophenol blue
    • 0.005% xylene cyanol
    • 0.5 mM EDTA
  • Uses:
    • Denaturing PAGE

Protocol:

  1. Add sample to an equal volume of RNA Loading Dye, (2X). Mix well.
  2. Heat at 65–70°C for 5–10 minutes to denature RNA.
  3. While heating the samples, setup the gel box and flush urea out of the wells with running buffer using a large tip.
  4. Load samples.