Agarose Gel Electrophoresis

Copyright Gel-Electrophoresis.com – Updated May 2011

Agarose gel electrophoresis is a separation method used in molecular biology research to separate out nucleic acids such as DNA and RNA by size.  The separation occurs in an electrical field which moves the charged molecules of DNA and RNA (both negatively charged due to their negatively charged sugar-phosphate backbone).  

Agarose Gel Migration Properties 

The molecules migrate through the agarose gel matrix and get separated by size.  The larger DNA or RNA molecules move slower and hence are closed to the loading wells.  Smaller nucleic acids are able to move quicker through the matrix and migrate the farthest away from the loading wells. 

This is in fact an over-simplification, as DNA can be super-coiled, nicked, single-stranded or double-stranded. Of these, super-coiled DNA usually migrates the fastest, followed by double-stranded, nicked, and then single-stranded if the size of the DNA is kept constant (i.e 1kb).

Factors in Agarose Gel DNA Migration  

Factors which influence the migration of DNA in agarose gel matrix include:

• size
• length
• confirmation
• supercoiling
• salt

For DNA migration, the length of the DNA is the most important factor influencing how far the DNA molecule will migrate. However, other factors also influence its migration including conformation of the DNA. Circular DNA found in plasmids (from bacteria), and also genomic DNA will exhibit altered migration, This is due to the fact that DNA is able to coil upon itself.  DNA in a coiled state is called supercoiled. Large supercoiled DNA migrates faster than its theoretical migration. This is due to a property called "Reptation" in which the supercoiled DNA "snakes length-wise" through the matrix.

Thus, supercoiled DNA moves fastest in an agarose matrix, whereas nicked DNA (or partly single stranded DNA) is less supercoiled and moves much slower than even linearized DNA. 

Agarose Gel Percentage and Resolving Power

Decreasing the matrix gaps by increasing the agarose concentration of a gel causes a further limit in the migration of molecules.  This enables the separation of smaller molecules. 

Voltage of Agarose Gel Electrophoresis 

Increasing the voltage, increases the electric current and causes molecules to migrate quicker through the matrix. The problem with this is that higher volages or currents increase the temperature of the agarose gel matrix. At high temperatures, the matrix expands first minimizing separation (usually causing streaking of samples) and later melts (you lose your samples!).

A rule is high voltages decrease the resolution (therefore do not use voltages higher 6 – 8 V/cm).

What is an Agarose Gel Composed of? 

An agarose gel is acually made usually by the researcher by using agarose. Dry agarose powder is mixed with water to a proper percentage (usually 1% agarose), and is then heated up (often in a microwave) in order to melt the agarose. Interestinly, agarose is a very pure form of agar made from a type of seaweed.

Applications of Agarose Gel Electrophoresis

Applications of agarose gels include:

Analytical Applications 

• Estimating the size of DNA or RNA molecules
• Estimating the amount and quality of DNA or RNA
• Analyzing restriction digestion fragments of DNA
• Analyzing PCR products from DNA amplification

Preparative Applications

• Separating restriction digested DNA prior to gel purification and subsequent cloning.
• Separation of DNA restriction fragments prior to Southern blot transfer
• Separation of RNA prior to Northern blotting and transfer.

Advantages and Disadvantages of Agarose Gel Electrophoresis 

Agarose gel electrophoresis has the following advantages:

• Agarose gels are quickly made and poured
• Agarose gels are relatively cheap
• Agarose gels are not run under denaturing conditions, therefore DNAs and RNAs (even proteins) are not denatured. Their activity is retained.
• Agarose gels are non-toxic adn clean (without ethidium bromide).
• Agarose gels are easier to handle than polyacrylamide-composed gels.
• Samples can also be recovered from agarose quite easily – see gel purification.

Agarose gel electrophoresis has the following disaddvantages: 

• Agarose gels melt during high voltage or current electrophoresis.
• Small fragments of DNA, RNA and small proteins are not resolved well on agarose gels.
• Nucleic acids of different properties including salt, coiling, and other properties run unpredictably.
• Gel running buffer can be exhausted.

 Originally posted – Copyright Gel Electrophoresis 2007

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