> For the complete documentation index, see [llms.txt](https://salis-lab.gitbook.io/protocol-book/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://salis-lab.gitbook.io/protocol-book/build-genetic-systems/dna/pcr-cleanup.md).

# PCR Cleanup

This protocol uses Zymo-Spin I Columns (product number: C1003-250) to recover ultra-pure DNA product from PCR, enzymatic reactions, and other sources. DNA can be eluted in as little as 5 uL and is ideal for DNA ligation, sequencing, labeling, PCR, microarray, transfection, transformation, restriction digestion, etc.

## Typical Applications

| Application                                     | Description                                                                                                                                       |
| ----------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------- |
| Post-PCR DNA Clean-up                           | Efficient desalting of DNA with the removal of DNA polymerases, primers and free dNTPs.                                                           |
| DNA Clean-up From Enzymatic Reactions           | Efficient desalting of DNA with the removal of modifying enzymes, RNA polymerases, ligases, kinases, nucleases, phosphatases, endonucleases, etc. |
| Post-Reverse Transcription (RT) & cDNA Clean-up | Efficiently purifies DNA following RT, either as a DNA/RNA complex or as single stranded cDNA following chemical hydrolysis of the RNA template.  |

## Buffer Preparation

* *Before starting*: Add X mL 100% ethanol to the Y mL **DNA Wash Buffer** concentrate. This step only needs to be comleted on first use. See bottle for volume of ethnanol to add. Mark the checkbox with an "X" where the bottle reads "Ethanol Added? \[]". If the bottle already has an "X", don't add ethanol!
* **DNA Wash Buffer** included is supplied ready-to-use and does not require the addition of ethanol prior to use.

## Protocol

*All centrifugation steps should be performed between 10,000 - 16,000 x g. We run our centrifuges at max, which is 13.2 x 1000 rpm.*

1. In a 1.5 mL microcentrifuge tube, add 2-7 volumes of **DNA Binding Buffer** to each volume of DNA sample (see table below). Mix briefly by vortexing.

| **Application**              | **DNA Binding Buffer : Sample** | **Example**     |
| ---------------------------- | ------------------------------- | --------------- |
| Plasmid, genomic DNA (>2 kb) | 2 : 1                           | 200 uL : 100 uL |
| PCR product, DNA fragment    | 5 : 1                           | 500 uL : 100 uL |
| ssDNA (e.g. cDNA, M13 phage) | 7 : 1                           | 700 uL : 100 uL |

1. Transfer mixture to a provided **Zymo-Spin Column** in a **Collection Tube**.
2. Centrifuge for 30 seconds. Discard the flow-through.
3. Add 200 uL **DNA Wash Buffer** to the column. Centrifuge for 30 seconds. Repeat the wash step.
4. Add >= 6 uL **DNA Elution Buffer** or water (we use water) directly to the column matrix and incubate at room temperature for one minute. Transfer the column to a 1.5 mL microcentrifuge tube and centrifuge for 30 seconds to elute the DNA.
5. (Optional) Measure the DNA concentration using the [Nanodrop](/protocol-book/build-genetic-systems/dna/pcr-cleanup.md).

Ultra-pure DNA is now ready for use.

## Note

This protocol is subject to change if Zymo Research improves the columns to be more efficient. The protocol described above was found [here](http://www.zymoresearch.com/downloads/dl/file/id/35/d4003i.pdf), but if this link is dead, or the product has been updated, just google for the new protocol. Make sure the correct product is specified - there are many different Zymo products for DNA purification.