DNA samples are mixed with DNA loading dye / buffer prior to loading into the wells of agarose gel for electrophoresis.
Usually a DNA loading dye contains at least one dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
EDTA and/or SDS can also added in the loading dye.
Ingredients are dissolved in water, but tris buffer can also be used in place of water. Tris containing DNA loading dye is called DNA loading buffer.
DNA loading dye/buffer serves the following purposes_ _ _ _
It provides high density to DNA sample. Due to high density, DNA sample settled at the bottom of the agarose wells. Nicely settled DNA form sharp and crisp band upon electrophoresis.
DNA loading dye/buffer imparts colour to DNA sample which allows easy monitoring of sample loading process. Sample overflow, cross-contamination, or leakage of wells can easily be monitored due to colour which otherwise would be very difficult with colourless sample.
Since dye color can be seen by naked eye, electrophoresis progression can be easily monitored by observing the migration of dyes which otherwise would be difficult because DNA migration can not be monitored by naked eye and DNA visualization requires staining and special instrument (ethidium bromide staining and uv torch).
There are several dyes (Bromophenol blue, Bromocresol green, Orange G, Xylene cyanol FF) which can be used for the preparation of DNA loading dye/buffer. An ideal dye, used for preparation of DNA loading dye/buffer, should have following characteristics.
It should not interact with the DNA or component of electrophoresis buffer and gel.
Like DNA, It should have negative charge and move towards the positive pole.
It should imparts contrast colour to DNA sample and its migration should easily be monitored visually.
It should not interfere with the analysis of DNA.
The size of the dye must be within the upper and lower resolution limit of the agarose gel (50 bp to 20 KB)
Two dye containing loading buffer (mostly bromophenol blue and xylene cyanol FF) is very common for DNA gel electrophoresis. In such loading buffer, One dye migrates fast and correspond to the migration of DNA fragment 50 – 500 bp, whereas other dye migrate comparatively slow and correspond to the migration of DNA fragment 4000 – 8000 bp.
Use of a specific dye mostly depends on the application and the size of the DNA fragment(s) to be analyzed by the gel electrophoresis. DNA band can be masked by co-migrating dye, which can give wrong information about the amount of DNA fragment. Depending on the size of DNA fragment, one should choose the dye.
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 sample settled at the bottom of the well. It also helps DNA sample to be confined in the well without diffusing out.
Since glycerol interacts with the borate of TBE electrophoresis buffer, glycerol containing loading dye / buffer are not recommended to use with TBE buffer.
Sucrose containing loading dye / buffer are prone to develop mold like growth. Therefore, sucrose containing loading dye / buffer can not be kept for long time at 4°C.
Ficoll-400 containing loading dyes / buffers can be stored at room temperature and also function as better density agent.
EDTA is added to inhibit the action of nucleases or DNA modifying enzymes which require divalent cation for their action.
Tris functions as a buffering agent and maintain the pH of the loading dye.
SDS reduces the DNA-protein interaction, thus helpful to run DNA samples containing proteins or enzymes (e.g., alkaline phosphatase).
DNA loading buffer is generally made in 6X concentration and mostly contains two dyes, bromophenol blue and xylene cyanol. Some DNA loading dyes contain only one dye while others may contain three dyes.