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Analysis of Hot Spring Microbial Mat Community by DGGE

Analysis of Hot Spring Microbial Mat Community by DGGE. Wed 7/8 1-1:30pm Pour gradient gels Read through protocols (Students will be divided into 2 groups) 1:30-2:30pm Lecture on DGGE 2:30-3pm Pour staking gels Prepare samples for loading 3-4pm Load samples!.

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Analysis of Hot Spring Microbial Mat Community by DGGE

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  1. Analysis of Hot Spring Microbial Mat Community by DGGE Wed 7/8 1-1:30pm Pour gradient gels Read through protocols (Students will be divided into 2 groups) 1:30-2:30pm Lecture on DGGE 2:30-3pm Pour staking gels Prepare samples for loading 3-4pm Load samples!

  2. Different microbial populations in a community DNA extraction and PCR amplification Mixed 16S rRNA gene copies Separate by cloning in E. coli or DGGE Phylogenetic identification Sequence

  3. Denaturing Gradient Gel Electrophoresis (DGGE) • Separate DNA fragments of the same length but with different sequences • Separation is based on the melting behavior of double-stranded DNA • Melting behavior depends on base-pair composition of the DNA

  4. DNA Structure Brock Biology of Microorganisms Figure 07-04

  5. Stronger! GC pairs > AT pairs Brock Biology of Microorganisms Figure 07-03

  6. Brock Biology of Microorganisms Figure 07-09 Denaturation of DNA = Melting ss DNA ds DNA Heat/Denaturant Melting temperature Function of the GC content of the DNA + Temperature Denaturant

  7. Denaturant (Formamide/Urea) 100% 0% Partially melted Separation Based on Differences in Nucleotide Sequence (G+C content) and Melting Characteristics Single strands Electrophoresis Or with GC-clamp Double strand

  8. PCR Amplification Mixed Population of DNA G+C-Tailed Product PCR Primers Separate on Denaturing Gradient Gel + G+C-Rich “Clamp” 16S rRNA Gene Denaturing Gradient Gel Electrophoresis B A C D Increasing Denaturant

  9. Hours 1.5 3 4.5 6 7.5 9 Time Travel DGGE Samples are loaded at regular intervals to determine optimum running time. Increasing Denaturant

  10. What can you know from DGGE? • Community complexity • Identify community members by sequencing • Distribution of microbial populations inhabiting different environments (e.g. temperatures) • Monitor community changes

  11. Example: Mushroom Spring Mat community A’ B A C 65°C 60°C 68°C 55°C 50°C

  12. DGGE Gel 1: Loaded by students A’ B A C 1 2 3 4 5 6 7 8 9 10 68 65 60 55 S1 S2 S3 S4 S5 S1 S2 S4 S5 Mushroom Spring S3 Mel

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