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To do downstream DNA sequence analysis after DGGE, the extraction of DNA from DGGE gel is a necessary step. DGGE or denaturing gradient gel electrophoresis is a molecular tool that is used to study the microbial community diversity. Though PCR amplified DNA sequences may have the same base-pair length they may vary in the GC content (i.e. sequence variants) and thus they may possess different melting points.
Different GC content (or DNA sequence variants) of the DNA fragments is the basis of their movements in the DGGE gel. In DGGE gel, DNA denaturants such as urea and formamide are added that interact with the hydrogen bonding among the nitrogen base pairs of the double stranded DNA fragments. For DGGE analysis, nested-PCR amplified DNA fragments are used.
These DNA fragments are separated in the DGGE gel based on their different melting points. After the DGGE run, DNA bands from the DGGE gel are excised under the UV trans-illuminator, and from the excised bands, DNA fragments are extracted.
For the extraction of DNA from DGGE gel, you are required to excise the bands from the gel and keep each band in a separate Eppendorf tube. Add 50 µl of TE buffer or sterile distilled water in each of the Eppendorf tube containing excised gel and incubate it overnight at 4 Â°C. You can also incubate it at room temperature for 2 hours. During incubation, DNA fragments come out of the DGGE gel and take out the supernatant in a new Eppendorf tube while leaving the gel particles.
The extraction of DNA from DGGE gel is necessary because components of the DGGE gel may affect the downstream PCR amplification. Now, extracted DNA is ready for the PCR analysis and downstream analysis. However, you should keep in mind that after DGGE, PCR amplification uses the same primers as used before DGGE, but this time, primers should not contain GC clamp.