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.

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, 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.