Agarose Gel Electrophoresis

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Agarose Gel Electrophoresis

This Agarose Gel Electrophoresis site is your portal for all things related to the agarose gel electrophoresis technique and its analysis. We provide you with all the background information, protocols, analysis steps, visualization and troubleshooting information for running agarose gels for DNA, RNA or Protein to become the ultimate expert in agarose gel electrophoresis!

Agarose Gel Electrophoresis Table of Contents

 

What is Agarose Gel Electrophoresis?

Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size however proteins can also be separated on agarose gels. Separation of the molecules is achieved by moving negatively charged nucleic acid molecules through an agarose matrix in an electric field (see electrophoresis). Shorter molecules move faster and migrate further than longer ones.

 

agarose gel electrophoresis

 

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis is mainly used in analysis or separation of DNA and RNA molecules (although proteins can also be separated on agarose gels), however separation also allows the purification of specific sizes of DNAs after restriction enzyme digestion. This is usually used in cloning to obtain cut plasmids, in which agarose gel electrophoresis separates cut vectors from uncut ones.

Thus agarose gels allow:

  1. Separation of restriction enzyme digested DNA including genomic DNA, prior to Southern Blot transfer. It is also often used for separating RNA prior to Northern transfer.
  2. Analysis of PCR products after polymerase chain reaction to assess for target DNA amplification.
  3. Allows for the estimation of the size of DNA molecules using a DNA marker or ladder which contains DNA fragments of various known sizes.
  4. Allows the rough estimation of DNA quantity and quality.
  5. Quantity is assessed using lambda DNA ladder which contains specific amounts of DNA in different bands.
  6. Quality of DNA is assessed by observing the absence of streaking or fragments (or contaminating DNA bands).
  7. Other techniques rely on agarose gel electrophoresis for DNA separation including DNA fingerprinting.

Advantages and Disadvantages of Agarose Gel Electrophoresis

The advantages are that the gel is easily poured, does not denature the samples. The samples can also be recovered.

The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.

Migration in Agarose Gels

The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of the DNA molecule is also a factor. To avoid this problem linear molecules are usually separated, usually DNA fragments from a restriction digest, linear DNA PCR products, or RNAs.

Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm).[citation needed]

Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid.

Resolving Limits of Agarose Gels

Agarose gels can be used for the separation of DNA fragments ranging from 50 base pairs to several megabases (millions of bases) by electrophoresis. More commonly however, agarose gel electrophoresis is used to separate DNA from PCR and cloning in the range of 100bp to about 15kb. Run times are about 30 minutes - 1 hour long.

Small DNAs or RNAs (smaller than 100bp) are better separated by polyacrylamide gels, however 2-3% agarose gels may be adequate to separate even 50bp fragments from much larger nucleic acids.

Recently however, it has been shown that up to one base pair size difference could be resolved on a 3% agarose gel with an extremely low conductivity medium such as 1 mM Lithium borate (Brody JR, Calhoun ES, Gallmeier E, Creavalle TD, Kern SE (2004). Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques. 37:598-602.).

 

Table of Agarose Gel Percentage and Efficient Range of Separation:

agarose gel percentage

As you can see low percentage agarose gels are best for the separation of large DNA molecules, whereas higher percentage gels are best for smaller DNAs.

 

DNA Purification with Agarose Gel Extraction

As mentioned before, agarose gel electrophoresis allows the separation and thus the purification of DNA for cloning. However, this usually requires high purity low melt agarose if the DNA is to be extracted from the gel.

Agarose Gel Electrophoresis Buffers

Depending on the size of the DNA electrophoresed and the application, different buffers can be used for agarose electrophoresis. TAE buffer (or Tris Acetate EDTA) is the most common used agarose gel electrophoresis buffer. TAE has the lowest buffering capacity of the buffers, however TAE offers the best resolution for larger DNA. However, TAE requires a lower voltage and more time.

However TBE buffer (Tris/Borate/EDTA) is often used for smaller DNA fragments (ie less than 500bp).

 

Sodium borate or SB buffer is a new buffer, however it is ineffective for resolving fragments larger than 5 kb. However SB has advantages in its low conductivity, allowing higher voltages (up to 35 V/cm). This could allow a shorter analysis time for routine electrophoresis.