• Analysis of DNA samples by agarose gel electrophoresis requires loading the samples onto the wells of agarose gel. The aqueous solution of DNA is colorless and has almost the same density as the electrophoresis buffer. When this DNA sample is loaded onto the wells of agarose gel, it does not remain in place and moves out of the wells due to diffusion as well as with the water current (the flow when a sample is pipetted out while loading). This results in loss of the DNA sample and the risk of contamination of neighboring wells. To solve this problem, DNA samples are mixed with DNA loading dye (also called sample loading dye) prior to loading onto the wells of agarose gel for electrophoresis. DNA loading dye not only makes the sample heavier and denser but also makes it coloured.
  
• DNA loading dye serves the following purposes_ _ _ _
  • It makes the DNA sample heavier and denser that helps the DNA sample to settle to the bottom of the agarose well without diffusing out, which results in sharp and crisp DNA bands on agarose gel after electrophoresis.
    
  • It makes the DNA sample coloured which allows easy monitoring of the sample loading process. Sample overflow, cross-contamination, or leakage of wells can easily be monitored due to color which otherwise would be very difficult with the colorless sample (watch the video DNA “Loading dye enable us to monitor the sample loading process”).
    
  • Tracking Dye, a component of the DNA loading dye, form bands that migrate towards the positive pole as DNA migrates. However, in contrast to DNA, the migration of these dyes can be seen by nacked eye that allows us to easily monitor the progression of electrophoresis without a need of special instruments and staining procedures (DNA visualization requires staining dyes and special instruments e.g., ethidium bromide staining and uv light).

• Although the composition and concentration of DNA loading dye can vary a lot, it essentially contains two components: Tracking dye and High-density reagent. Several tracking dyes (such as bromophenol blue, xylene cyanol, orange G etc) and high-density reagents (glycerol, sucrose, Ficoll 400 etc) are suitable for the preparation of DNA loading dye. One can choose them depending on specific requirements and availability of reagents.
  
• The most simple DNA loading dye can contain one tracking dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high-density reagent (glycerol, sucrose or Ficoll 400). DNA loading dye composition as simple as Bromophenol blue-saturated solution of Glycerol [Take 1 ml Glycerol (Glycerol conc ≥ 85%) in 1.5 ml eppendorf, add a pinch of bromophenol blue, vortex vigorously and centrifuge at high speed, use supernatant and let the undissolved bromophenol pellet at the bottom] can serve as DNA loading dye. EDTA and/or SDS can also be added in the loading dye. Ingredients are dissolved in water, but the Tris buffer can also be used. Tris-containing DNA loading dyes are called DNA loading buffers.
• The most common loading dyes contain two tracking dyes (Xylene cyanol and bromophenol blue or Xylene cyanol and Orange G) and a high-density reagent (glycerol, sucrose or Ficoll 400). In such a loading dye, one tracking dye migrates relatively fast and corresponds to the migration of small DNA fragments ranging from 50 – 500 bp, whereas other dye migrates comparatively slowly and corresponds to the migration of large DNA fragments (4000 – 8000 bp). DNA loading dyes can be prepared or purchased as a 6x or 10x concentrated solution.
• The choice of a specific tracking dyes primarily depends on the size of the DNA fragment(s) to be analyzed by the gel electrophoresis. Tracking dyes that comigrate with the DNA fragments are avoided as they can mask the DNA band. Masked DNA bands are not visible properly when viewed under UV light, resulting in wrong interpretation of the experiment.
• The high-density reagent (glycerol, sucrose, or Ficoll 400) is added to facilitate the sample to be placed in the well of the gel. Due to high density, DNA samples settle at the bottom of the well. It also helps DNA samples to be confined in the well without diffusing out.
• The choice of high-density reagent depends on the availability of reagents, storage and other reagents used for agarose gel electrophoresis. For example, glycerol-containing DNA loading dyes are not recommended with TBE electrophoresis as glycerol is known to interact with the borate (Sciarra & Elliott, 1960). Sucrose-containing DNA loading dyes are prone to mold-like growth even at 4°C. Ficoll 400-containing loading dyes can be stored at room temperature and also function as a better density agent, but the cost of Ficoll 400 may limit its use.
• EDTA is added in the DNA loading dye to inhibit the action of nucleases or DNA modifying enzymes that require divalent cation for their action.
• Tris functions as a buffering agent and maintains the pH of the loading dye.
• SDS reduces DNA-protein interactions, thus helpful to run DNA samples containing proteins or enzymes (e.g., alkaline phosphatase).

Tracking dyes enable us to monitor progress of electrophoresis

In this video, you can see the migration of tracking dye in agarose gel.

DNA Loading dye enable us to monitor the sample loading process

The sample colour due to tracking dyes of DNA loading dye helps you to locate the sample distribution while loding onto wells of agarose gel and any mishappening due to any error leading to loss of sample or contamination of local wells.

REFERENCES

• Sciarra & Elliott, 1960. A solubility study of the boric acid-glycerin complex. I. Solubility of boric acid in glycerin solution at 25 degrees. J Am Pharm Assoc Am Pharm Assoc. 49, 115-117. PMID-13854731; Full Text Links: wiley, readcube, booksc