Included in the kit are control reagents and primers to detect human ribosomal protein gene, RPL30, and a 16s RNA universal region in E. coli DNA, as well as a sample of human IMR-90/E. coli (10:1 ratio) DNA. (Sequences of the primers and target DNA are listed on the manual pages 16–17). A typical control reaction that will yield a sufficient quantity of eluted material to perform an agarose gel assay or qPCR reactions at least in triplicate is outlined below:
- Perform capture experiment using 2.5 μl (250 ng) of IMR90/E. coli DNA as described on the manual pages 5–7.
- Carefully transfer the supernatant containing the target microbial DNA to a fresh microcentrifuge tube, and perform AMPure Bead Clean-up or Ethanol Precipitation. This is your enriched microbial DNA sample.
- Wash bead pellet (Step 2 in "Collect Enriched Microbial DNA" Section) and elute the DNA by incubation at 75°C in 50 μl of TE plus Proteinase K, as described here .
- Carefully remove the supernatant to a fresh microcentrifuge tube, and perform AMPure Bead Clean-up or Ethanol Precipitation. This is your host DNA sample.