View source for 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).

==Material==

[[Image:Agarose gel dyes.jpg|right|150px]]

===Density===
* Ficoll
* sucrose
* glycerol

===Colour===

{| cellpadding=5 style="border: 1px solid #CC9;" 
 ! 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
 |
 |}

<nowiki>*</nowiki>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
* 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===
*[[Knight:Loading dye]]

== Links ==
=== OWW ===
* [[Agarose gel electrophoresis]]
* [[Cresol Red]]

=== External ===
* Methodsbook.net: [http://www.methodbook.net/dna/agarogel.html#loadingbuff xylene or bromophenol + sucrose DNA loading buffers] [[Image:2stars.png]]
* 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]]