Agarose gel loading buffer - OpenWetWare
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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
1 Material
1.1 Density
1.2 Colour
2 Recipes for loading buffers
2.1 Ficoll & Orange G (6×)
2.2 Sucrose & xylene cyanol / bromophenol blue (6×)
2.3 Glycerol & bromophenol blue (6×)
2.4 NEB Loading Dye
2.5 Specific recipes
3 Links
3.1 OWW
3.2 External
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
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Agarose gel loading buffer - OpenWetWare
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https://openwetware.org/wiki/Agarose_gel_loading_buffer
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] (http://www.cshprotocols.org/cgi/content/full/2006/7/pdb.rec8658?ijkey=df90f0
b5bc3257b067b31a4943c953b1fd4b55db&keytype2=tf_ipsecsha&text_only=true) (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.
6×
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
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Agarose gel loading buffer - OpenWetWare
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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
0.02% Orange G
1.2 mg/mL
Specific recipes
Knight:Loading dye
Links
OWW
Agarose gel electrophoresis
Cresol Red
External
Methodsbook.net: xylene or bromophenol + sucrose DNA loading buffers (http://www.methodbook.ne
t/dna/agarogel.html#loadingbuff)
Bioron Company page on bromophenol + xylene + Ficoll DNA loading buffer (http://www.bioron.net/
products/dna-and-dna-markers/loading-buffer/)
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This page was last edited on 24 February 2016, at 06:37.
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