Molecular Biology Topics

TIPS FOR PREPARATION OF AGAROSE GEL

Written by MicroDok

Agarose is simply defined as a polysaccharide material or powder that can be used to form a gel to separate molecules (e.g. DNA molecules) according to their individual sizes. This type of gel used for separation of DNA molecules based on sizes is generally known as agarose gel. Agarose is a polysaccharide that is extracted from certain seaweeds. Agarose gel is a unique type of gel or reagent required for performing gel electrophoresis technique in the molecular biology laboratory. This gel is prepared from agarose powder after following certain protocol. Other types of gels such as polyacrylamide gel also exist for performing molecular / cell biology experiments. But only the preparation of agarose gel electrophoresis shall be highlighted in this section. Agarose gel is an important reagent in the molecular biology laboratory because it allows scientists to separate DNA fragments on the basis of their sizes by electrophoresis technique. No agarose gel no electrophoresis technique. Fragments of DNA molecules can be separated by electrophoresis through an agarose gel. For the separation of very small fragments, an acrylamide gel is usually used. The agarose gel is
stained with ethidium bromide dye, and when the DNA samples is applied to the wells or slots in the gel, a voltage is applied to across the gel, and the DNA fragments. This leads to the formation of an ethidium bromide–DNA complex that fluoresces when exposed to an ultraviolet light. The migration of a DNA fragment in an agarose gel is usually determined by its size. Generally, larger DNA fragments move more slowly through the agarose gel while smaller DNA fragments move faster. DNA is negatively charged, and will therefore move towards the positive electrode or anode when placed in an electric field. Since smaller DNA fragments move faster than larger DNA fragments, gel electrophoresis can be used to estimate the sizes of specific DNA fragments. The agarose gel electrophoresis technique is one of the easiest ways of separating and analyzing DNA molecules after carrying out polymerase chain reaction (PCR).

Tips for preparation of agarose gel for gel electrophoresis technique

  • 1 % agarose gel means: 1 g agarose powder in 100 ml distilled water + 1 X TBE. This implies that you should measure or weigh out 1 g of agarose powder and dispense or pour same into 1 X TBE buffer. TBE = tris borate EDTA buffer
  • 5 % agarose gel means: 1.5 g agarose powder in 100 ml distilled water + 1 X TBE
  • 100 % agarose gel means: 100 g agarose powder in 100 ml distilled water + 1 X TBE
  • It is noteworthy that in the preparation of TBE buffer, you have to prepare tris base separately. Also prepare boric acid separately. Then mix tris base + boric acid + EDTA solution to get TBE buffer.
  • 10 X means 1 liter (1000 ml)
  • 1 X means 100 ml

The preparation of agarose gel does not require sterilization / autoclaving as is the case when preparing culture media in the microbiology laboratory. Therefore distilled water is required for the preparation of agarose gel required for gel electrophoresis technique. After the proper mixing of the agarose powder, distilled water and the TBE buffer, the solution is mixed thoroughly and heated in a microwave oven until it dissolves properly by boiling. After this, pour the solution in to the casting tray of the electrophoretic tank, and allow the gel to solidify. Ensure to place the comb prior to pouring the molten agarose gel into the casting tray of the electrophoretic tank. The comb helps to form slots or wells in the gel, where you will insert the DNA samples to be analyzed. Remove the combs gently when the gel is casted and solidified.

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