Agarose gel loading buffer: Difference between revisions
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==Material== | ==Material== | ||
[[Image:Agarose gel dyes.jpg|right|150px]] | |||
===Density=== | ===Density=== | ||
* Ficoll | * [[Ficoll]] | ||
* sucrose | * sucrose | ||
* glycerol | |||
===Colour=== | ===Colour=== | ||
| Line 13: | Line 16: | ||
! 0.5-1.5% agarose | ! 0.5-1.5% agarose | ||
! 2.0-3.0% agarose* | ! 2.0-3.0% agarose* | ||
! CAS number | |||
! order # example | ! order # example | ||
|- | |- | ||
| | | Xylene cyanol | ||
| 4000 | | 10'000-4000 bp | ||
| 750 bp | | 750-200 bp | ||
| 2650-17-1 | |||
| Sigma X4126 | | Sigma X4126 | ||
|- | |- | ||
| | | [[Cresol Red]] | ||
| 400- | | 2000-1000 bp | ||
| | | 200-125 bp | ||
| 62625-29-0 | |||
| Sigma 114480 | |||
|- | |||
| Bromophenol blue | |||
| 500-400 bp | |||
| 150-50 bp | |||
| | |||
| Sigma B8026 | | Sigma B8026 | ||
|- | |- | ||
| Line 28: | Line 40: | ||
| <100 bp | | <100 bp | ||
| ? | | ? | ||
| | | | ||
| Sigma O3756 | |||
|- | |||
| Tartrazine | |||
| <20 bp | |||
| <20 bp | |||
| 1934-21-0 | |||
| | |||
|} | |} | ||
<nowiki>*</nowiki>sieving agarose | <nowiki>*</nowiki>sieving agarose | ||
==Recipes== | ==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. | 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 (6x) === | ||
*Ficoll 400 | * 1.5g Ficoll 400 | ||
* Orange G dye | |||
*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°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=== | ===Sucrose & xylene cyanol / bromophenol blue (6x) === | ||
* 4g sucrose | * 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]] | ||
== Links == | == Links == | ||
=== OWW === | === OWW === | ||
* [[Agarose gel electrophoresis]] | * [[Agarose gel electrophoresis]] | ||
* [[Cresol Red]] | |||
=== External === | === External === | ||
* http://www.methodbook.net/dna/agarogel.html#loadingbuff | * 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]] | |||
Revision as of 11:20, 15 July 2012
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
Density
- Ficoll
- sucrose
- glycerol
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
- Methodsbook.net: xylene or bromophenol + sucrose DNA loading buffers
- Bioron Company page on bromophenol + xylene + Ficoll DNA loading buffer
