WENDY CHAO'S LABORATORY PROTOCOLS

GUIDE TO LOADING BUFFERS AND TRACKING DYES

My loading buffer of choice contains Ficoll-400 (for density), orange G, and xylene cyanol. Get the recipe here.

Why this loading dye is superior:

1. The high molecular weight Ficoll-400 stays at the bottom of the well - unlike sucrose or glycerol which diffuse quickly - thus yielding sharper DNA bands.

2. Ficoll can be stored at room temperature, whereas glycerol and sucrose should be refrigerated. Furthermore, sucrose can get moldy or otherwise yucky.

3. Glycerol can interact with the borate in TBE gels, altering local pH.

4. This loading dye contains Orange G - instead of bromophenol blue (BPB) - in addition to xylene cyanol (XC). It results in a pretty green color than separates into blue and orange - much more interesting than the regular BPB/XC combination. Commonly used dyes are listed below.

5. BPB truly sucks anyway - it is very dark and obscures DNA bands. In agarose gels, BPB typically runs in the 200-700 bp range (see gel mobility chart) - precisely where your PCR fragments are likely to be, or where the smaller restriction fragments are faint and diffuse.

6.Orange G runs approximately with the smallest detectable DNA fragments in agarose gels (around 50 bp), which is a better indicator of when your DNA is about to run off the gel.

Dyes (color, relative weight in 1% agarose):

• Xylene cyanol (light blue, 4000 bp)
• Cresol Red (lipstick red, 1000 bp)
• Bromophenol blue* (dark blue, 400 bp)
• Orange G (school bus orange, 50 bp)

See also dye mobility chart

*Bromophenol blue truly sucks - it's dark and obscures DNA bands in the 200-700 bp range, precisely where PCR bands usually are, or where smaller restriciton fragments are faint and diffuse.

Dye mobility chart

For xylene cyanol (XC) and bromophenol blue (BPB) only. I plagiarized this from somewhere, I just can't remember where. Just know that cresol red runs between XC and BPB, and that orange G runs around 50 bp.