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
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
