PCR Purification using silica-membrane-based column purification
Updated 5/3/2019 10:48 PM
Introduction
Materials
- Equipment
- Microcentrifuge capable of holding 1.5mL tubes and producing at least 17,900 x g
- Sterile 1.5mL microcentrifuge tubes
- Pipettes for pipetting volume ranges from 1 μL to 1000 μL
- Sterile pipette tips
- ______ PCR Purification kit
- Spin Columns
- Collection tubes
- Binding and wash buffers
- Elution Buffer or alternative
Procedure
Important information to be documented throughout this procedure
- Samples’ names and storage location (if applicable)
- Volume of PCR sample used
- What PCR Purification kit was used
- Batch number of your kit
- Unexpected and expected observations and results (either in writing or in picture form)
- Clarification notes (protocol modifications, issues, significant notes)
Important Notes
- This protocol was designed after Qiagen's QIAQuick PCR Purification Kit protocol. If the kit used has a set of directions that differ significantly, follow the manufacturer's protocol and note changes.
- Verify that ethanol (96–100% purity) has been added to wash buffer before use (see bottle label for volume)
Wash buffer that has had ethanol added should be clearly marked as such with a black X on the top, with a date of addition written.
- All centrifugation steps are carried out at 17,900 x g (rcf) in a conventional table-top microcentrifuge at room temperature
- Always ensure the centrifuge is balanced properly
- Elution of pure DNA with ≤50µL requires the buffer to be added directly to the center of the membrane
Procedure
- In a sterile 1.5mL microcentrifuge tube, add 5 volumes binding buffer to 1 volume of the PCR reaction and mix. Spin down if necessary.
For example, add 500µL of binding buffer to 100µL PCR sample.
- Place a silica column in a provided 2mL collection tube.
CRITICALMake sure to label the silica columns.
- To bind DNA, apply the sample to the silica column and centrifuge for 1 min.
- Discard flow-through. Place the silica column back into the same collection tube.
- To wash, add 750µL wash buffer to the silica column and centrifuge for 1 min. Discard flow-through and place the silica column back into the same collection tube.
- Centrifuge the silica column once more for 1 min to remove residual wash buffer.
This step ensures any residual ethanol, which may interfere with subsequent enzymatic reactions, is removed.
- Properly label a sterile 1.5mL microcentrifuge tube with sample details, your initials and date. Place the silica column from the previous step into the labelled tube.
- To elute DNA, add 30µL elution buffer (10mM Tris·Cl, pH 8.5) to the surface of the silica membrane, let the column stand for 2 min and centrifuge for 1 min.
Storage + Disposal
- If the purified DNA is NOT to be immediately used, temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box. Properly note where it was stored.
- The columns should be placed in the tube rack next to the sink for used PCR Purification columns. Used microcentrifuge tubes can be disposed in lab trash.
This protocol describes the method used for silica-membrane-based column purification of up to 10µg PCR products (100bp to 10kb in size) using the _____ PCR Purification Kit. The purified DNA may subsequently be used in downstream assays, including sequencing, microarray analysis, ligation and transformation, restriction digestion, labeling, microinjection, PCR, and in vitro transcription.
DNA adsorbs to the silica membrane in the spin column in high-salt conditions and a pH ≤7.5 provided by the binding buffer (eg. buffer PB in a QIAquick kit). Impurities such as primers, unincorporated nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide and dyes are washed away by the ethanol-containing wash buffer (eg. buffer PE in a QIAquick kit). Pure DNA is eluted under low-salt and basic pH conditions with a small volume of elution buffer (eg. buffer EB in a QIAquick kit).