DNA Sequencing and Fragment Analysis

An interesting article was posted March 25, 2011 on BitesizeBio.com titled 5 Ways to Destroy Your Agarose Gel. Every researcher may have made some of these common mistakes at one time. The five ways provided are…

  1. Use water instead of buffer for the gel or running buffer.
  2. Forget to add ethidium bromide
  3. Use the wrong percentage (or type) of agarose.
  4. Switch the leads from the power source.
  5. Drop the gel on the way to the imager.

The focus of this article is to explain the importance of using the correct percentage gel. In many genetic analysis applications a 1% agarose gel is commonly used to test plasmid preparations and PCR fragments. However, the resolution of the 1% gel may not sufficiently resolve smaller DNA products.

Percent Agarose Determines Pore Size

Agarose gel electrophoresis is a form of chromatography. The gel provides the stationary phase and electrical current provides the mobile phase. Charged particles such as DNA will migrate towards the positively charged anode in response to an electrical current across the gel. The gel provides the resistance against DNA migration. Smaller fragments move more rapidly than larger fragments.

Resistance is directly proportional to the porous nature of an agarose gel. Smaller pores provide more resistance. Increasing the percent of the gel decreases the size of the pores. When the pore sizes are too large small DNA fragments migrate together and do not become separated (figure 1). This figure illustrates why large DNA fragments should not be run on an agarose gel with small fragments of DNA.

Correct Percent of Agarose Depends on the Size Products Tested

The correct percent agarose gel is dependant on the size of the fragment that will be tested. Plasmid DNA preparations that are 5 kb to7 kb resolve well on a 1% gel. Large PCR fragments that are similar in size to plasmid DNA could also resolve on a 1 % percent gel. However, small PCR fragments that require smaller pore size for better resolution require a higher percent gel. General guidelines for mixing the correct percent gel are provided in table 1.

For small PCR fragments less than 500 bases in size, it is best to use a two percent gel. This will increase the run time. However, it will also improve resolution of fragments that are similar in size and may not resolve on lower percentage gels.




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