Agarose gel loading buffer

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(Specific recipes)
 
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==Material==
==Material==
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 +
[[Image:Agarose gel dyes.jpg|right|150px]]
===Density===
===Density===
-
* Ficoll
+
* [[Ficoll]]
* sucrose
* sucrose
 +
* glycerol
===Colour===
===Colour===
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* xylene cyanol (Sigma X4126)
 
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* bromophenol blue (Sigma B8026)
 
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* Orange G
 
-
==Recipes==
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{| cellpadding=5 style="border: 1px solid #CC9;"
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! dye
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! 0.5-1.5% agarose
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! 2.0-3.0% agarose*
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! CAS number
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! order # example
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|-
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| Xylene cyanol
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| 10'000-4000 bp
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| 750-200 bp
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| 2650-17-1
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| Sigma X4126
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|-
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| [[Cresol Red]]
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| 2000-1000 bp
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| 200-125 bp
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| 62625-29-0
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| Sigma 114480
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|-
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| Bromophenol blue
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| 500-400 bp
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| 150-50 bp
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|
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| Sigma B8026
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|-
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| Orange G
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| <100 bp
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| ?
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|
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| Sigma O3756
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|-
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| Tartrazine
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| <20 bp
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| <20 bp
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| 1934-21-0
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|
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|}
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===Ficoll & Orange G ===
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<nowiki>*</nowiki>sieving agarose
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*Ficoll 400
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*Deionized water
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*Orange G dye
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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&deg;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.
+
==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 (6x) ===
 +
* 1.5g Ficoll 400
 +
* Orange G dye
 +
* dH<sub>2</sub>O to 10mL
 +
 
 +
Add very small amounts of Orange G dye such that the loading dye is dark orange.  Store in small aliquots at 4&deg;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) ===
 +
* 4g sucrose
 +
* 25mg bromophenol blue or xylene cyanol (0.25%)
 +
* dH<sub>2</sub>O 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 (6x) ===
 +
* 3ml glycerol (30%)
 +
* 25mg bromophenol blue (0.25%)
 +
* dH<sub>2</sub>O to 10mL
 +
 
 +
compare CSH protocols [http://www.cshprotocols.org/cgi/content/full/2006/7/pdb.rec8658?ijkey=df90f0b5bc3257b067b31a4943c953b1fd4b55db&keytype2=tf_ipsecsha&text_only=true] (restricted access)
===Specific recipes===
===Specific recipes===
*[[Knight:Loading dye]]
*[[Knight:Loading dye]]
-
==Use==
+
== Links ==
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*Orange G: generally runs very fast (<100 bp)
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=== OWW ===
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*Bromophenol blue: purple, generally runs at ~500bp (depending on percentage agarose)
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* [[Agarose gel electrophoresis]]
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*Xylene cyanol: blue, runs at ~4kb
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* [[Cresol Red]]
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=== External ===
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* Methodsbook.net: [http://www.methodbook.net/dna/agarogel.html#loadingbuff xylene or bromophenol + sucrose DNA loading buffers] [[Image:2stars.png]]
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* Bioron Company page on [http://www.bioron.net/products/dna-and-dna-markers/loading-buffer/ bromophenol + xylene + Ficoll DNA loading buffer] [[Image:2stars.png]]
 +
 
 +
 
 +
[[Category:Material]] [[Category:Agarose gel electrophoresis]]

Current revision

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 (6x)

  • 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 (6x)

  • 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 (6x)

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

compare CSH protocols [1] (restricted access)

Specific recipes

Links

OWW

External

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