Bead clean up ratios used in the Illumina 16S demonstrated protocol

Below are the bead clean-up ratios used after each PCR step of the Illumina 16S protocol.

PCR Clean‐Up 1

• This step uses purification beads to purify the 16S V3 and V4 amplicon away from free primers and primer dimer species.

• 25 µl of samples + 20 µl beads = 0.8X beads:DNA ratio

PCR Clean‐Up 2

• This step uses purification beads to clean up the final library before quantification.

• 50 µl of libraries + 56 µl beads = 1.12X beads:DNA ratio

Note: As the beads:DNA ratio changes, the length of fragments binding to the beads and left in solution also changes--the lower the ratio of beads:DNA, the larger the final fragments will be at elution.

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