Method of TGGE
To separate nucleic acids by TGGE, the following steps must be performed:
- Preparing and pouring the Gels - Because a buffered system must be chosen, it is important that the system remain stable within the context of increasing temperature. Thus, urea is typically utilized for gel preparation; however, researchers need to be aware that the amount of urea used will have an impact on the overall temperature required to separate the DNA (Biometra, 2000).
- Electrophoresis - The gel is loaded, the sample is placed on the gel according to the type of gel that is being run—i.e. parallel or perpendicular—the voltage is adjusted and the sample can be left to run (Biometra, 2000). Depending on which type of TGGE is to be run, either perpendicular or parallel, varying amounts of sample need to be prepared and loaded. A larger amount of one sample is used with perpendicular, while a smaller amount of many samples are used with parallel TGGE.
- Staining – Once the gel has been run, the gel must be stained to visualize the results. While there are a number of stains that can be used for this purpose, silver staining has proven to be the most effective tool (Biometra, 2000).
- Elution of DNA – the DNA can be eluted from the silver stain for further analysis through PCR amplification (Biometra, 2000).
Read more about this topic: Temperature Gradient Gel Electrophoresis
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