Staining¶

Common dyes¶

Bromophenol blue¶

|150 - 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¶

|150 - 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

SYBR¶

SYBR SAFE
- Theoretical sensitivity: 500 pg
SYBR™ Gold
- Theoretical sensitivity: 25 pg; picks up ssDNA too
- Practical sensitivity (clear band): 10 ng

Procedure¶

Fill a new tray with some buffer from the electrophoresis tray and transfer the gel to it.
Staining: Add 5 μL of a dye and wrap the tray with tin foil or place in a covered box (dyes are often light sensitive).
Place on a rocker for 15-30 min at ~15 rpm.
Destain the gel by washing it a few times with dH2O and leaving in dH2O for a few minutes.
- This step improves gel clarity even for SYBR Gold.
Image the gel:
- GelDoc device in the 3rd floor lab, or
- Typhoon in the hallway:
  - Choose you dye from the drop-down menu in the Fluorescence panel (typically, SYBR Gold)
  - Start with 400 V.
  - If you cannot see anything or the image is in thew range, increase the voltage (up to 1000V), which increases laser's power. If still nothing is visible, edit the protocol to use Bi-Alkali regime (instead of)
  - Conversely, if everything is saturated or in the high range, decrease the voltage.
  - Pixel size is linked to imaging speed. Lower pixel size gives you a better resolution but a longer scan time.

Troubleshooting¶

Wide streaks instead of narrow bands: sample overloaded
Large blobs with shades of dye (after imaging):
- Stain not properly distributed during gel preparation, i.e., it was not stirred into the gel enough
- Consider destaining by washing the gel a few times with dH2O and then storing it in dH2O (covered from light) for 1 h.