# 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](https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf).

**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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