Agarose gel loading buffer

Loading dye is mixed with DNA samples for use in agarose gel electrophoresis. It generally contains a dye to assess how "fast" your gel is running and a reagent to render your samples denser than the running buffer (so that the samples sink in the well).

Material

Density

• Ficoll
• sucrose

Colour

• xylene cyanol (Sigma X4126)
• bromophenol blue (Sigma B8026)
• Orange G

Recipes

The precise amount of dye is not important. However, it is crucial that you prevent overlay of dye and expected DNA size. For example, if you are expecting a genotyping band of 200-400nt, you shouldn't use bromophenol blue since it will obscure your product. In this case, you should use a larger dye like xylene cyanol.

Ficoll & Orange G

• Ficoll 400
• Deionized water
• Orange G dye

Dissolve 1.5 g of Ficoll in 10 mL of deionized water. Add very small amounts of Orange G dye such that the loading dye is dark orange. Store in small aliquots at 4°C (room temperature is okay too). To use, add and mix 1/5th volume of loading dye to DNA solutions prior to loading into the wells of gels.

Sucrose & xylene cyanol / bromophenol blue

• 25mg bromophenol blue or xylene cyanol
• 4g sucrose
• H₂O to 10mL

Store at 4°C to avoid mould growing in the sucrose. 10mL of loading buffer will last for years.

Specific recipes

• Knight:Loading dye

Use

• Orange G: generally runs very fast (<100 bp)
• Bromophenol blue: purple, generally runs at ~500bp (depending on percentage agarose)
• Xylene cyanol: blue, runs at ~4kb