Agarose gel loading buffer

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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).

Contents

Material

Density

Colour

dye 0.5-1.5% agarose 2.0-3.0% agarose* CAS number order # example
Xylene cyanol 10'000-4000 bp 750-200 bp 2650-17-1 Sigma X4126
Cresol Red 2000-1000 bp 200-125 bp 62625-29-0 Sigma 114480
Bromophenol blue 500-400 bp 150-50 bp Sigma B8026
Orange G <100 bp  ? Sigma O3756
Tartrazine <20 bp <20 bp 1934-21-0

*sieving agarose

Recipes for loading buffers

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 (6×)

  • 1.5g Ficoll 400
  • Orange G dye
  • dH2O to 10mL

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 (6×)

  • 4g sucrose
  • 25mg bromophenol blue or xylene cyanol (0.25%)
  • dH2O 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.

Glycerol & bromophenol blue (6×)

  • 3ml glycerol (30%)
  • 25mg bromophenol blue (0.25%)
  • dH2O to 10mL

compare CSH protocols [1] (restricted access)

NEB Loading Dye

Used by Dueber Lab (UC Berkeley). –Shyam Bhakta
Ficoll®-400 results in brighter and tighter bands when compared to glycerol loading dyes. SDS creates sharper bands, as some restriction enzymes are known to remain bound to DNA following cleavage. EDTA chelates magnesium (up to 10 mM) in enzymatic reactions, thereby stopping the reaction. Tris-HCl buffers the sample at a pH safe for DNA. In 1% agarose gels, orange G comigrates with a ~50 bp fragment, bromophenol blue with a ~300 bp, and xylene cyanol with a ~4,000 bp fragment. This 6× loading dye recipe is identical to that of NEB loading dyes, except for the addition of both xylene cyanol and Orange G (slightly reduced from 0.15%) with bromophenol blue. The Dueber Lab also adds 6× GelGreen DNA stain to the loading dye, so as not to have to pre/post-stain the gel.

1× (reference)
per mL
Either 60% v/v Glycerol 0.6 mL 100% glycerol 10% Glycerol
or 15% m/v Ficoll®-400 150 mg 2.5% Ficoll®-400
20 mM Tris-HCl, pH 8.0 20 mM÷cstock 3.33 mM Tris-HCl, pH 8.0
60 mM EDTA 60 mM÷cstock 10 mM EDTA
0.48% m/v SDS 0.48%÷cstock. Or 4.8 mg/mL. 0.08% SDS
0.03% m/v Xylene Cyanol 0.3 mg 0.005% Xylene Cyanol
0.03% m/v Bromophenol Blue 0.3 mg 0.005% Bromophenol Blue
0.12% m/v Orange G 1.2 mg/mL 0.02% Orange G

Specific recipes

Links

OWW

External

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