Agarose gel loading buffer

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(local lab specific info removed)
(Sucrose & xylene cyanol / bromophenol blue recipe)
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==Recipes==
==Recipes==
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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 & Orange G ===
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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.
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.
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===Sucrose & xylene cyanol / bromophenol blue===
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* 25mg bromophenol blue or xylene cyanol
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* 4g sucrose
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* H<sub>2</sub>O to 10mL
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Store at 4°C to avoid mould growing in the sucrose. 10mL of loading buffer will last for years.
===Specific recipes===
===Specific recipes===

Revision as of 07:16, 20 April 2007

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

  • 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
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