Recipes for loading buffers
The actual quantity of dye matters not. However, it is vital that you prevent overlay of dye and expected DNA size. For instance, if you're expecting a genotyping gang of 200-400nt, you should not use bromophenol blue because it will obscure your products. Within this situation, you need to use a bigger dye like xylene cyanol.
Ficoll Orange G (6)
Add very small quantities of Orange G dye so that the loading dye is dark orange. Store in small aliquots at 4C (70 degrees is ok too). To make use of, add and blend 1/fifth amount of loading dye to DNA solutions just before loading in to the wells of gels.
Sucrose xylene cyanol / bromophenol blue (6)
- 4g sucrose
- 25mg bromophenol blue or xylene cyanol (.25%)
- dH2 O to 10mL
Add appropriate add up to DNA sample, e.g. 5µl to 25µl.
Store at 4°C to prevent mould growing within the sucrose. 10mL of loading buffer will last a long time.
Glycerol bromophenol blue (6)
- 3ml glycerol (30%)
- 25mg bromophenol blue (.25%)
- dH2 O to 10mL
compare CSH protocols [1] (restricted access)
NEB Loading Dye
Utilized by Dueber Lab (UC Berkeley). –Shyam Bhakta
Ficoll®-400 leads to better and tighter bands in comparison with glycerol loading dyes. SDS creates sharper bands, as some restriction enzymes are recognized to remain certain to DNA following cleavage. EDTA chelates magnesium (as much as 10 mM) in enzymatic reactions, therefore stopping the response. Tris-HCl buffers the sample in a pH safe for DNA. In 1% agarose gels, orange G comigrates having a
50 bp fragment, bromophenol blue having a
300 bp, and xylene cyanol having a
4,000 bp fragment. This 6 loading dye recipe is similar to that particular of NEB loading dyes, aside from adding both xylene cyanol and Orange G (slightly reduced from .15%) with bromophenol blue. The Dueber Lab also adds 6 GelGreen DNA stain towards the loading dye, so they won't need to pre/publish-stain the gel.
