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).
|dye||0.5-1.5% agarose||2.0-3.0% agarose*||order # example|
|xylene cyanol||4000-5000 bp||750 bp||Sigma X4126|
|bromophenol blue||400-500 bp||100 bp||Sigma B8026|
|Orange G||<100 bp||?||?|
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 (6x)
- 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 (6x)
- 25mg bromophenol blue or xylene cyanol
- 4g sucrose
- H2O to 10mL
Add appropriate amount to DNA sample, e.g. 5µl to 25µl.
Store at 4°C to avoid mould growing in the sucrose. 10mL of loading buffer will last for years.
- Methodsbook.net: xylene or bromophenol + sucrose DNA loading buffers
- Bioron Company page on bromophenol + xylene + Ficoll DNA loading buffer