“A dye used to monitor the migration of DNA into a gel or during gel electrophoresis is known as DNA gel loading dye.”

Loading dye is an important component in agarose gel electrophoresis. The loading dye comprises bromophenol blue, Ficoll 400 and water majorly while Xylene cyanol, Tris and EDTA are optional in it.

Bromophenol blue is one of the most popular indicators of DNA in agarose gel electrophoresis. Bromophenol blue is a pH indicator. It is a weak acid and available as a light pink to a purple crystal and water-soluble. 

It is even used as a color indicator, acid-base pH indicator and as a biological stain. At pH 3 it will give a yellow color and pH above 4.8 it will give a blue color.

Name:                      bromophenol blue

Chemical name:     3′,3′′,5′,5′′-Tetrabromophenolsulfophthalein sodium salt

Chemical formula: C₁₉H₉Br₄NaO₅S

Abbreviation:          BPB, loading dye

Molecular weight:  691.94

The function of gel loading dye:

Gel loading dye has three important functions in agarose gel electrophoresis:

It is utilized as a color indicator to monitor the migration of DNA in gel electrophoresis. See, DNA is colorless and odorless, we can’t see its migration in a gel. Thus we need some chemicals that can migrate above it. So that we can stop it running out of the gel.  Electrophoresis progression can be monitored by using the BPB (bromophenol blue).

DNA is less dense and hence it diffuses in a running buffer. We need to settle it on the bottom of the well. The loading dye contains Ficoll or glycerol that gives density to the DNA sample. Henceforth, DNA can’t come out and diffuse in the buffer.

It makes DNA settle on the bottom of the well. The settled DNA can migrates properly and gives nice and sharpened bands on to gel.

BPB runs parallel to 100bp to 300bp in 0.8% agarose gel, 150bp in 2% agarose gel, and 50bp in 3% agarose gel concentration, so it runs ahead of the DNA fragment. Because of this, DNA migration can be strictly monitored. 

Read another article of gel electrophoresis series: Role of EtBr in molecular genetics and cytogenetics.

We can also monitor sample overflow, cross-contamination or leakage due to the colour composition of loading dye. 

Read more:

1. DNA sequencing
2. 10 tips on how to do PCR
3. Importance of Tris-EDTA (TE) buffer in DNA extraction

The composition of dye is very important because the clarity of the result will be obtained by maintaining the proper concentration of components used in the loading dye.

DNA gel loading dye composition:

The composition of 1X loading dye,

• 0.042 % (W/V) Bromophenol blue powder
• 2.5 % Ficoll
• 0.042% (W/V) Xylene cyanol FF (optional)
• Final makeup with D/W

To make an effective DNA gel loading dye, the loading dye should have the following characteristics:

• It can increase the effectiveness and quality of the result, another dye called a Xylene Cyanol and should be used in combination with the Bromophenol blue.

• The dye should impart contrast color and can migrate ahead of DNA. If it migrates with our DNA fragment than the fragment cannot be visualized properly under UV light.

• The dye contains a negative charge (more specifically the BPB) so that it can migrate toward the positive node in agarose gel electrophoresis.

• The component of the gel loading dye should not interact with DNA otherwise it can affect the structural hierarchy and mobility of the DNA.

• Further, It cannot react with agarose.

• The loading dye must be temperature stable. Sometimes, the temperature of the agarose buffer will increase at a high voltage. In this situation, the components of loading dye must remain stable otherwise the gel melted and we can’t get good results.

• The stability of the loading dye should be longer.

The recipe for loading dye varies depending upon the experience of the researcher. Some scientist adds EDTA and Tris in loading dye too. 

Read related articles: Importance of Tris-EDTA (TE) buffer in DNA extraction.

The EDTA inhibits the nuclease activity and Tris maintains the pH of the loading dye. However, these ingredients are optional because EDTA and Tris are already present in the electrophoresis buffer.

The Ficoll 400 is highly recommended over other polysaccharides. Generally, glycerol is avoided for the gel loading dye because it can react with the borate in the TBE buffer. This will decrease the clarity of the result. Nonetheless, the role of glycerol in electrophoresis is the same as Ficoll 400.

Sucrose can also not recommended because the sucrose-containing dye cannot be stored for a longer time. Hence Ficoll 400 is the best choice for making gel loading dye.

Ficoll 400 is a hydrophilic, higher molecular weight, polysaccharide which is non-reactive with DNA, buffer or agarose.

Related article: Agarose gel electrophoresis.

My ultimate guide for using DNA loading dye effectively:

Always make 10X gel loading dye and store it in 10 different aliquots.

• 0.42 % (W/V) Bromophenol blue powder
• 25 % Ficoll
• 0.42% (W/V) Xylene cyanol FF (optional)

This is your 10X DNA loading dye composition. Add water as per your requirement.

We can also prepare a 6X loading dye. Ordinarily, the ready to use loading dye comes in 6X concentration.

6X loading dye preparation:

• 0.25% (W/V) bromophenol blue
• 0.25% (W/V) xylene cyanol FF
• 15% (W/V) Ficoll 400
• add water as per requirement

From the stock solution of the 6X gel loading dye, firstly, we have to prepare to work 1X dye and then we can use it for electrophoresis.

It is better to add DNA gel loading dye directly to the PCR tubes containing our PCR amplicon. A 10μl sample can be loaded into the agarose gel well. So for 25μl of PCR product roughly add 5 to 7μl of DNA gel loading dye to the PCR tubes. 

Now you can take 10μl directly from the tubes and load it into the well. Do not forget to mix it properly before loading.

If possible use ready to use mastermix which contains the loading dye already into it. This will save you time. Takara is one of the best master mix, which I prefer to use.

If your experiment is sensitive and crucial, do not use loading dye containing mastermix. If your PCR product is further will be used for restriction digestion or sequencing, do not add loading dye directly into the PCR tubes. Instead, follow another way.

Take a parafilm strip and place it on the bench. Put a small drop of loading dye on it (7μl) and add the PCR product into the dye.

Read other articles:

• What are the different components used in the PCR reaction buffer?
• Importance of Tris-EDTA (TE) buffer in DNA extraction

Mix well and load it into the gel. However, this technique is time-consuming and the chance of cross-contamination is always higher.

Always use high-quality ingredients and chemicals for the best results and also, weigh each chemical properly. 

You can also use other dye like bromocresol green, Orange G or Xylene cyanol FF.

Conclusion:

Bromophenol blue is one of the best choices for the DNA gel loading dye preparation. However, the ready to use DNA mastermix containing dye performs well too.

The bromophenol blue is one of the widely used dye in agarose gel electrophoresis of DNA. You can make a stock and store it to use for a long time.