DNA gel loading dye composition, preparation and function.
Loading dye is an important component in agarose gel electrophoresis. The loading dye contains bromophenol blue, Ficoll 400 and water majorly while Xylene cyanol, Tris and EDTA are optional.
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. It is a water soluble.
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It is even used as a colour indicator, acid-base pH indicator and as a biological stain. At pH 3 it will give yellow colour and pH above 4.8 it will give a blue colour.
Name: bromophenol blue
Chemical name: 3′,3′′,5′,5′′-Tetrabromophenolsulfophthalein sodium salt
Chemical formula: C19H9Br4NaO5S
Abbreviation: BPB, loading dye
Molecular weight: 691.94
Function of gel loading dye:
Gel loading dye has three important functions in agarose gel electrophoresis:
- It gives the colour indication for migration of DNA. Electrophoresis progression can be monitored due to the BPB.
- The loading dye gives density to the DNA. DNA solution is less dense, hence it comes out from the well. The loading dye contains the Ficoll or glycerol which gives density to the DNA. Therefore DNA cannot come out from the well.
- It settled DNA into the bottom of the well. The settled DNA can migrates properly and gives nice and sharpened bands on to gel.
BPB runs parallel to 1000bp 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 does not run out from agarose gel.
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We can also monitor sample overflow, cross-contamination or leakage because of the colour composition of loading dye.
The composition of dye is very important because the clarity of the result will be obtained by maintaining the proper concentration of component used in loading dye.
DNA gel loading dye compositon:
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 loading Dye, the loading dye should have the following characteristics:
- To increase the effectiveness and quality of the result, another dye called as Xylene Cyanol should be used in combination with the Bromophenol blue.
- The dye should impart contrast colour and can migrate ahead of DNA. If it migrates with our DNA fragments 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 dye should not interact with DNA otherwise it can affect the structural hierarchy and mobility of DNA.
- Further, It should not 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 situations, the components of loading dye remain stable otherwise it will hinder in the result.
- The shelf life of 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.
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The EDTA inhibits the nuclease activity and Tris maintains the pH of 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 TBE buffer. This will decrease the clarity of result. Nonetheless, the role of glycerol in electrophoresis is same as Ficoll 400.
Sucrose can also not recommended because sucrose containing dye cannot be stored for longer time. Hence Ficoll 400 is the best choice for making lading dye.
Ficoll 400 is a hydrophilic, higher molecular weight, polysaccharide which is non-reactive with DNA, buffer or agarose.
My ultimate guide to use DNA loading dye effectively:
Always make 10X 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 6X loading dye. Generally, 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 6X gel loading dye, firstly we have to prepare working 1X dye and then we can use it for electrophoresis.
It is better to add DNA loading dye directly to the PCR tubes which contain our PCR amplicon. Generally, a 10μl sample can be loaded into the agarose gel well. So for 25μl of PCR product roughly add 5 to 7μl of DNA loading dye to the PCR tubes.
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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 your 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 PCR product into the dye.
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.
Article written by: Tushar Chauhan
Article reviewed by: Tushar Kachhadiya