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  • Downstream Analysis (E2612)

    Quantitation of DNA by agarose gel electrophoresis

    1. Prepare the unenriched sample by diluting 0.1 μg of unenriched input DNA in TE Buffer for a final volume of 20 μl.

    2. Aliquot 20 μl of the unenriched DNA, supernatant fraction containing the enriched mircobial DNA, and eluted fraction containing the eukaryotic host DNA into separate tubes. Add 5 μl of Gel Loading Dye, Blue (6X) (NEB #B7021) or other loading buffer to each sample.

    3. Load 20 μl of each sample alongside an appropriate DNA marker (λ DNA-HindIII Digest, NEB #N3012) on a 0.8% agarose gel. Since the supernatant fraction containing enriched microbial DNA will be depleted of host DNA, the total amount of DNA seen on the gel will be reduced compared to the input control.
      Agarose gel containing unenriched, enriched and captured host DNA
      Lane M, Lambda DNA-Hind III Digest, NEB #N3012; Lane 1, Unenriched input DNA; Lane 2, Supernatant fraction containing enriched microbial DNA; Lane 3, Eluted DNA from magnetic bead pellet containing host DNA.

    Validation of enrichment by qPCR

    1. Prepare the unenriched (input) sample by diluting 1 μg in TE Buffer for a final volume of 50 μl.

    2. Aliquot 5 μl in triplicate of each sample (input, enriched, beads/pellet) for the human RPL30 gene and the 16S rRNA gene qPCR reaction.

    3. Add 0.5 μl of the appropriate primer set for a 20 μl qPCR reaction volume.

    4. Perform qPCR using the following program:
      5 minutes at 95°C
      10 seconds at 95°C
      30 seconds at 60°C
      40 cycles.

    5. Analyze quantitative PCR results using software provided with the realtime PCR machine.

    6. Little to no change in the Ct value of the 16S RNA E. coli target should be observed between the input and supernatant fractions. A 6–7 Ct drop should be observed between the input and supernatant fraction in the RPL30 target.