Oct 04, 2025
Purification
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11752vr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 04, 2025
Protocol Integer ID: 228969
Keywords: qiagen dna purification kit, purification pcr product purification, purification, pcr, dna
Abstract
PCR product purification using the QIAGEN DNA Purification Kit.
Materials
- QIAquick PCR Purification Kit
- PCR reaction sample (20 µL)
- Benchtop centrifuge (capable of ≥13,000 rpm)
- Microcentrifuge tubes (1.5 mL)
- Pipettes and tips
Troubleshooting
Purification
Mix PCR product with binding buffer
Add 20 µL of PCR reaction mixture into a clean 1.5 mL microcentrifuge tube. Then add 100 µL of Buffer PB (5× the PCR volume). Mix thoroughly with a pipette. Do not centrifuge.
Load the mixture onto the purification column
Carefully transfer the entire mixture onto the center of a QIAquick spin column placed inside a 2 mL collection tube. Ensure all liquid is applied directly to the center of the membrane.
First centrifugation and discard flow-through
Centrifuge at 13,000 rpm for 1 minute. Discard the flow-through collected in the tube, but keep the spin column.
Wash the silica membrane
Add 750 µL of Buffer PE (pre-diluted with 96–100% ethanol according to the manual) to the spin column. Centrifuge at 13,000 rpm for 1 minute.
Remove residual ethanol
Discard the flow-through and place the column back into the empty collection tube. Centrifuge again at 13,000 rpm for 1 minute to completely remove residual ethanol, which could interfere with downstream applications.
Elute DNA
Transfer the spin column to a clean 1.5 mL microcentrifuge tube. Add 30 µL of Buffer EB directly to the center of the membrane (avoid touching the column walls). Let stand at room temperature for 1 minute to allow DNA release from the silica membrane.
Final centrifugation and collection
Centrifuge at 13,000 rpm for 1 minute to collect the eluate. The flow-through contains the purified PCR product.