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Electrophoresis Dyes

Posted by Anonymous on Fri, 2015-12-04 17:06

Electrophoresis Dyes: Hi, We have recently purchased our own equipment for doing
electrophoresis and would like to know what the best dyes to use are and their preparation
please?

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Year Level:
Senior Secondary
Laboratory Technicians:
Laboratory Technicians

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Electrophoresis Dyes
Submitted by sat on 11 December 2015

In Brief

The electrophoresis of food dyes and scientific stains in school science laboratories
demonstrates the basic principles and procedures of gel electrophoresis in a simple way. The
dyes and stains used are affordable, safe, and the procedure simpler than using DNA. A big
advantage is that they behave in a similar way to DNA molecules during electrophoresis, and
any bands produced can be visualized directly in the gel, without any further staining.

Different scientific stains produce distinct bands of colour during electrophoresis, whilst food
dyes can be separated into their different pigments.

Samples of stains or food dyes are required to be mixed with a 20% v/v glycerol/distilled water
solution prior to electrophoresing. The glycerol will make the sample denser than the
electrophoresis buffer and allow the sample to sink and remain in the well rather than float into
the buffer. This is an important step, so that no dye floats into the buffer and cross
contaminates other samples.

Samples can be loaded into the gel using micropipettes, transfer or Pasteur pipettes. Care
needs to be taken not to puncture the bottom of the well or overfill the wells. Loading every
second well is also a good idea to minimise the chance of contamination between adjacent
wells. A 10–20 uL (1drop) sample size is generally used and the samples are electrophoresed
at 90–100 volts for 30–35 min.

Preparation of samples

Food dyes can be purchased at the supermarket. They are negatively charged molecules like
DNA molecules and will migrate to the anode (+ve terminal).

Preparation of food dyes for electrophoresis

1. Prepare 2 mL of a 20% v/v glycerol/distilled water solution.

2. Using a transfer or automatic pipette, add 0.1–0.2 mL of food dye to the glycerol solution
and mix thoroughly until the dye is evenly distributed.

3. Repeat steps 1 and 2 for each colour.

4. Store the prepared dye solutions at 4° C for a longer shelf life.

Electrophoresis of food dyes will result in the following pigment separations.

 Green = blue and yellow bands

Red = pink and red/orange bands

Yellow = pink, orange and yellow bands

Blue = light blue, dark blue and dark red bands

Black = orange/red, yellow, orange, pink, light blue and dark blue

Scientific stains such as Bromophenol Blue, Methyl Green, Orange G, Indigo Carmine, Rose
Bengal, Tartrazine and Xylene Cyanol are commonly used. These can be purchased from
various scientific suppliers as ready-to-use reagents, or can be made up from individual
powdered stains at a concentration of 0.2% w/v in 20% glycerol/distilled water. Stains can be
used as a mixture, often referred to as a ‘ladder’. They produce distinct bands of colour when
electrophoresed.

Most scientific stains are negatively charged and will migrate towards the anode (+ve
terminal). Methyl Green is positively charged and will migrate towards the cathode (-ve
terminal). The smaller the molecule the further it will migrate.

Preparation of stain solutions for electrophoresis

1. Prepare 10 mL of a 20% v/v glycerol/distilled water solution.

2. Add 20 mg of the required stain to the glycerol solution and mix thoroughly until the
powder has completely dissolved. This may take a while.

3. Store the prepared stain solution in a brown bottle at room temperature to increase shelf
life (slowing decomposition).

4. If requiring a mixture, then combine prepared stains in a 1:1 ratio for use.

Some safety and other considerations

The power supply and the gel apparatus use high voltage and should be handled with
caution. Ensure electrodes are in place before turning on. Make sure the power supply
 is turned off before disconnecting any leads and when not in use.

Do not operate the power supply in damp or humid conditions. Keep water away from
the power source.

Gloves and eye protection should be worn at all times during the procedure.

Observe the position of the bands during the electrophoresis procedure, so that it can be
stopped when sufficient separation has occurred and to make sure that the bands do not
run off the end of the gel.

Measurements and photographs should be taken soon after stopping the

electrophoresis as the molecules will continue to diffuse into the gel.

If using samples that will migrate to both the anode and cathode, the wells should be
prepared in the centre of the gel to allow good visualisation of the stains as they move to
the different electrodes.

At the end of the experiment, place all used gels in a plastic bag and dispose in the
general waste bin. Used buffer solutions can be disposed of down the sink.

Safety Data Sheets (SDSs) should be consulted as a first step in assessing the risks
associated with a chemical: most of the stains used during electrophoresis are irritants.

Additional information

Gel electrophoresis

Gel electrophoresis is the separation of molecules through their migration in an electric field.
The molecules are separated based on their size, electrical charge and shape when applied
to a gel medium. Electrophoresis is a commonly used technique in laboratories to analyse
DNA, RNA and protein samples, plus other compounds in mixed samples. The principle of
electrophoresis is that molecules are placed into a gel that is subjected to an electric current.
Any negatively charged molecules will move towards the anode (+ve terminal) , whilst
positively charged molecules will move towards the cathode (-ve terminal). The smaller the
molecule, the further it will migrate. The density of the gel and the type of buffer used will
affect how a molecule migrates. Most biological molecules are electrically charged, causing
them to move when subjected to an electric field.

Types of gels for electrophoresis

There are several different types of agar that can be used to separate food dyes and scientific
stains. Mobility of molecules is affected by gel concentration. Higher percentage gels, as well
as thicker gels, are sturdier and easier to handle but the migration of molecules will take
longer. Gels can be prepared, poured and stored at 4° C for several days before use.

Type of gel Concentration Use

Most widely used to

separate DNA.

Molecular biology
Agarose: a polysaccharide
grade gives the best
derivative of agar.
results
1% w/v in buffer solution
Can be used for stains
and food dyes.

Gives the best

resolution.

Can be used for stains

and food dyes.

Does not produce gels

with uniform pore sizes
Agar for microbiological media 1% w/v in buffer solution which will influence the
electrophoretic process.

Bands will be more

diffuse.
 Can be used for stains
and food dyes.

Does not produce gels

with uniform pore sizes
Agar from health food stores 1% w/v in buffer solution which will influence the
electrophoretic process.

Bands will be more

diffuse.

Edited 19 February 2020: Correction: The gel should not be made up with water. The gels
should be made up in the same buffer used to run the gel in the electrophoresis chamber.
This results in all the ions equilibrating in both the gel and the running buffer resulting in nice
sharp bands. If using two different buffer types you run the risk that you end up with blurred
and warped bands.

Buffers

The buffer solution is used to conduct the electric current and maintain the pH during the
process. A buffer is poured into the electrophoresis chamber, filling the wells until it just
covers the top of the casting tray. There are several different types of buffers that can be used
to separate dyes and stains. The most commonly used buffer solutions are TAE (Tris-acetate-
EDTA) and TBE (Tris-borate-EDTA) and are quite expensive. However, a safer and cheaper
option for schools is to use a 10 mM sodium bicarbonate buffer.1, 2

References

Edited 19 February 2020 to remove/update links that are no longer valid.

1 ‘Dyes for Electrophoresis’, Southern Biological website,
https://www.southernbiological.com/dyes-for-electrophoresis/ (Updated February 2020)

2 ‘Getting started with Gel Electrophoresis’, Southern Biological website,

https://www.southernbiological.com/getting-started-with-gel-electrophore... (Updated February
2020)

‘Activity 2: Gel Electrophoresis of Dyes’, The American Phytopathological Society (APS)

website, (Accessed December 2015) https://www.apsnet.org/Pages/default.aspx and search
for ‘gel electrophoresis’ (Updated February 2020)

‘Basic Biotechnology Kit Gel electrophoresis of dyes’, University of California Davis website,
https://pbge.faculty.ucdavis.edu/wp-content/uploads/sites/408/2016/10/Ge... (Updated
February 2020)

Bio-Tek Services Pty Ltd. n.d. Agarose Gel Electrophoresis using dyes – Instruction manual,
Biotechnology Education website,
https://web.archive.org/web/20120321125959/http://www.biotechnologyeduca... (Internet
Archived Version, April 2018)

Carolina Biological Supply Company, 2005. Introductory Gel Electrophoresis – Teacher’s

Manual with Student Guide, Centre for Precollegiate Education and Training, University of
Florida website https://web.archive.org/web/20150513210641/http://www.cpet.ufl.edu/wp-
content/uploads/2013/10/Intro-Gel-Electrophoresis-manual.pdf (Original resource no longer
available. This version provided by the Internet Archive October 2019)

Flinn Scientific Inc. 2008. Dyeing for Electrophoresis, Flinn Scientific Inc. website,
https://www.flinnsci.com/media/452802/bf10901.pdf Further resources can be found at Flinn
Scientific, visit https://www.flinnsci.com/, search for electrophoresis and then select the
resources tab, see also ‘Introduction to Electrophoresis’ and ‘Basic Electrophoresis’. (Updated
February 2020)

‘Forensic Science: Building Your Own Tool for Identifying DNA’ Science Buddies website,
https://www.sciencebuddies.org/science-fair-projects/project-
ideas/BioChem_p028/biotechnology-techniques/forensic-science-building-your-own-tool-for-
identifying-dna (Updated February 2020)

Queensland Museum. 2006. Food dye electrophoresis – Student task sheet, Queensland
Museum website,
https://www.qm.qld.gov.au/microsites/qx/pdf/dd002_food_dye_electrophoresis.pdf

Southern Biological, n.d. Gel Electrophoresis of Dyes, Southern Biological website,

https://www.southernbiological.com/biotechnology/biotech-consumables/g42-90-dyes-for-
electrophoresis/ (Updated October 2019).

Safety data sheets:

Chem-Supply. 2014. Bromophenol Blue Safety Data Sheet. Search

https://www.chemsupply.com.au/ to source the latest Safety Data Sheet via the product
information page.

Chem-Supply. 2013. Tartrazine Yellow (C.I.19140) Safety Data Sheet. Search

https://www.chemsupply.com.au/ to source the latest Safety Data Sheet via the product
information page.

Chem-Supply. 2011. Indigo Carmine, Safety Data Sheet. Search

https://www.chemsupply.com.au/ to source the latest Safety Data Sheet via the product
information page.

Sigma-Aldrich. 2013. Rose Bengal, Safety Data Sheet, Search

https://www.sigmaaldrich.com/australia.html to source the latest Safety Data Sheet via the
product information page.

Sigma-Aldrich. 2013. Xylene Cyanol FF, Safety Data Sheet. Search

https://www.sigmaaldrich.com/australia.html to source the latest Safety Data Sheet via the
product information page.

Electrophoresis Dyes
Submitted by KelliS on 08 December 2015

We found it easier to buy the dyes from Southern Biological.

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