# 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.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #6127), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000006127%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-faq-list/000006127.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.