Electrophoresis Lab Report

Electrophoresis – An Introduction

• An analytical technique in which the motion of scattered particles run through a fluid under the influence of uniformly charged field is called electrophoresis.

• This phenomenon was first observed by Ferdinand Frederic Reuss followed by Arne Tiselius who won the Nobel Prize in chemistry for his research on electrophoresis, adsorption analysis and his discoveries concerning the complex nature of serum protein. It is a technique used in laboratories in order to separate macromolecules based on molecular size and charge.

• This technique applies a negative charge so proteins move towards a positive charge. This is used for both nucleic acids and proteins. Nucleic acids like DNA and RNA, while proteins

However, different molecules will move at quite different and individual rates depending on the physical characteristics of the molecule and on experimental system used. The velocity of movement, ν, of a charged molecule in an electric field depends on variables described by
  Eq/ f
• Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules travel toward anode (positive) pole. The migration flow is resulted solely by the molecular weight where small weight molecules migrate faster than larger ones. In addition to size separation, nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes expunging the desired “band” from a stained gel viewed with a UV transilluminator.

• In order to visualize nucleic acid molecules in agarose gels, ethidium bromide or SYBR Green are commonly used dyes. Illumination of the agarose gels with 300-nm UV light is subsequently used for visualizing the stained nucleic

The resolution of the DNA changes with the percentage concentration of the gel. Increasing the agarose concentration of a gel reduces the migration speed and improves separation of smaller DNA molecules, while lowering gel concentration permits large DNA molecules to be separated. For a standard agarose gel electrophoresis, a 0.7% gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1k fragments

• They are the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation but a relatively low resolving power, since the bands formed in the gels tend to be fuzzy and spread apart.

• This is a result of pore size and cannot be largely controlled. These and other advantages and disadvantages of using agarose gels for DNA electrophoresis are summarized in Table below :

Advantages Disadvantages
• Gels are quick and easy to cast
• Nontoxic gel medium
• Good for separating large DNA molecules
• Can recover samples by melting the gel, digesting with enzyme agarose • Poor separation of low molecular weight samples
• High cost of agarose Fuzzy