Purification of PCR fragments from agarose gels
Purification of PCR fragments from agarose gels
The Wizard SV Gel and PCR Purification System is a method for the direct purification of PCR fragments from agarose gels or from PCR amplification reaction mixtures using an in silico method. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Purification of PCR fragments from agarose gels
Materials and Instruments
Ethanol Ethidium bromide Electrophoresis buffer PCR fragments Agarose gel Cells and tissues Move I. Materials For more product details, please visit Aladdin Scientific website.
Microcentrifuge tubes Dissecting knives UV lamps Vacuum units
1. Buffers, solutions and reagents
(1) B-enzyme (95%)
(2) Embedded dyes such as ethidium bromide
(3) TAE electrophoresis buffer
40 mmol/L trimethylolaminomethane-acetate, pH 8.2
1 mmol/LEDTA
or (4) TBE electrophoresis buffer
90 mmol/L trimethylolaminomethane-borate, pH 8.3
2 mmol/LEDTA
2. Nucleic acids and oligonucleotides
PCR fragments from agarose gels
3. Gel
Agarose gel (regular or low melting point)
4. Specialized equipment
Microcentrifuge tube, 1.5 ml
Dissecting knife or razor blade
Long wavelength UV lamp
Vacuum unit and vacuum adapter (Promega; for vacuum purification)
5. Other
WizardSV gel and PCR purification system (Promega; includes SV microcolumns, collection tubes, membrane binding solution, membrane washing solution, and nuclease-free water)
6. Cells and Tissues
PCR Samples for Purification
II. Methods
Standard safety clothing should be worn, especially when handling agarose gels stained by ethidium bromide, with gloves and a UV-shielding face shield to protect the eyes and face from UV light.
1. Dissolution of the gel
(1) Place the PCR samples onto the lanes of the agarose gel and electrophoreze according to established methods.
(2) Weigh and record the weight of the 1.5 ml microcentrifuge tube used to store the gel.
(3) Use a long-wavelength ultraviolet lamp and a dye such as ethidium bromide to image the DNA and take a picture. To minimize the formation of DNA nicks, irradiate the condensed limb for as short a time as possible (GrundemannandSchomig1996;Zimmermanneta!.1998). The agarose gel containing the target DNA fragments is cut off using a clean scalpel or razor blade, and the volume of the condensed limb is kept as small as possible. The gel was transferred to a weighed microcentrifuge tube, weighed and the mass recorded, and the mass of the empty tube was subtracted from the total mass to obtain the mass of the gel block.
(4) Add membrane binding solution at a rate of 10ul of solution per 10 mg of agarose gel.
(5) Shake the mixture and incubate at 50-65°C for l0 min until the gel is completely dissolved, shaking the tube every few minutes to increase the rate of agarose gel melting, and centrifuging the tube briefly at room temperature to ensure that the contents are at the bottom of the tube. Once melted, the agarose gel will not re-solidify at room temperature.
(6) DNA can be purified by centrifugation or vacuum filtration.
2. Centrifugation for DNA Purification
(1) Place an SV microcolumn in the collection tube corresponding to each target fragment.
(2) Transfer the dissolved gel mixture to the SV microcolumn and hold at room temperature for lmin.
(3) Centrifuge at 1000g for 1min, remove the microcolumn, discard the liquid in the collection tube, and return the microcolumn to the collection tube.
(4) Wash the microcolumn by adding 700ul of membrane washing solution to the microcolumn and centrifuging the microcolumn at 10000 g for lmin. Empty the collection tube as described above, put the microcolumn back into the collection tube and repeat the washing with 500ul of Membrane Wash Solution and centrifuge at 10,000g for 5 min.
(5) Carefully remove the microcolumn to avoid wetting the bottom of the column with filtrate. If the column becomes wet, empty the collection tube and centrifuge again for 1 min.
(6) Transfer the microcolumn to a clean 1.5 ml microcentrifuge tube and add 50 ul of nuclease-free water directly to the center of the column without touching the pipette tip to the membrane. Hold at room temperature for 1 min and centrifuge at 10,000 g for 1 min.
(7) Discard the microcolumn and store the eluted DNA at 4°C or -20°C.
3. Vacuum purification of DNA
(1) Attach a vacuum adapter with a Luer-Lok device to the port of the vacuum unit corresponding to each gel or PCR sample containing the target fragment, and insert an SV microcolumn into each vacuum adapter so that it fits snugly into its empty adapter.
(2) Transfer the dissolved clot mixture or PCR amplification reaction mixture to the microcolumns and incubate at room temperature for 1 min, pushing the liquid completely through the microcolumns by evacuating.
(3) Wash the microcolumn by adding 700ul of Membrane Wash Solution to the microcolumn, making sure that all droplets on the microcolumn wall from the previous step are thoroughly washed away, and after pushing the liquid through the microcolumn completely by evacuating, perform a second wash with 500ul of Membrane Wash Solution.
(4) Stop the evacuation and open an unused port to remove the vacuum unit. Remove the microcolumn from the vacuum unit, transfer it to a collection tube, and centrifuge at l0000 g for 5 min to remove all residual Flag Wash Solution.
(5) Transfer the microcolumn to a clean 1.5 ml microtube and add 50ul of nuclease-free water directly to the center of the column without touching the membrane. Hold at room temperature for 1 min and centrifuge at 10000 g for 1 min.
(6) Discard the microcolumn and store the eluted DNA at 4°C or -20°C.