Recovery of DNA fragments from polyacrylamide gels (crush and soak method)
Recovery of DNA fragments from polyacrylamide gels (crush and soak method)
The standard method for recovering DNA from polyacrylamide gels is the "crush and soak" technique first introduced by Maxam and Gilbert (1977). The eluted DNA is usually free of enzyme inhibitors and contaminants that are toxic to cell transfection or microinjection. This method takes a long time, but the amount of work is small. The recovery rate is less than 30% to 90%, depending on the size of the DNA fragments. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Recovery of DNA fragments from polyacrylamide gels (crush and soak method)
Principle
The standard method for recovering DNA from polyacrylamide gels is the "crush and soak" technique first introduced by Maxam and Gilbert (1977). The eluted DNA is usually free of enzyme inhibitors and contaminants that are toxic to cell transfection or microinjection. This method takes a long time, but the amount of work is small. The recovery rate is less than 30% to 90%, depending on the size of the DNA fragments.
Materials and Instruments
DNA Standard Reference DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Acrylamide gel elution buffer Chloroform Ethanol Gel loading buffer Phenol Chloroform Sodium acetate TE
Polyacrylamide gel Single-use plastic column Rotor or rotating platform UV light
1. Buffers and solutions
Acrylamide gel elution buffer (0.5 mol/L magnesium acetate, 10 mmol/L magnesium acetate tetrahydrate, 1 mmol/L EDTA (pH 8.0), 0.1% (m/V) SDS (optional or not))
Chloroform
Ethanol
6X gel loading buffer
Phenol: Chloroform (1:1, V/V)
Sodium acetate (3 mol/L, pH 5.2)
TE (pH 8.0)
2. Gel
Polyacrylamide gel
Polyacrylamide gels and high resolution agarose gels of appropriate concentration
3. Nucleic acids and oligonucleotides
DNA standard reference, obtained by restriction enzyme digestion of a known amount of DNA sample
DNA samples
4. Specialized equipment
Single-use plastic chromatography columns (e.g., Quik-Sep columns, Isolabs, Inc.) or syringe cartridges with Whatman GF/C filter-paper-dense siliconized glass wool
Rotating wheel or rotating platform in a 37°C incubator
Portable long-wave (302 nm) UV lamp
Methods
1. Perform polyacrylamide gel electrophoresis of DNA samples and standard reference material according to the protocol "Neutral Polyacrylamide Gel Electrophoresis". Detect the gel stained with ethidium bromide or SYBR Gold by radioactive autoradiography or long-wave UV lamp (302 nm) to determine the location of the target DNA.
2. Cut the gel containing the target band with a clean, sharp scalpel or razor blade, making the cut polyacrylamide gel strip as small as possible. This can be done in any of the following ways:
(1) Cut the gel with the Saran Wrap membrane under ultraviolet light and peel the gel strip from the membrane.
(2) Illuminate the gel from below with a UV lamp and outline the location of the DNA strip on the back of the glass plate with a permanent marker (e.g., Sharpie pen). Turn the gel over, remove the Saran Wrap membrane, and cut the DNA strip according to the markings made by the marker.
(3) When detecting by autoradiography, place the exposed autoradiography film on the Saran Wrap membrane and align it with the gel. Use a marker to outline the location of the desired DNA fragment on the back of the glass plate. Remove the film and Saran Wrap membrane and cut the strip.
3. Transfer the cut gel strip into a microcentrifuge or polypropylene tube and squeeze the gel against the wall of the tube with a disposable pipette tip or inoculating needle.
Alternatively, use a clean scalpel or razor to cut the gel into small strips before placing it into the elution tube.
4. Estimate the approximate volume of the gel strip and add 1 to 2 times the volume of Acrylamide Gel Elution Buffer to the microcentrifuge tube.
5. Cover the tube and incubate at 37°C on a rotating wheel or rotating platform.
6. Centrifuge the sample in a bench-top centrifuge at maximum speed for 1 min at 4°C. Transfer the supernatant to a new centrifuge tube, being careful not to transfer any of the polyacrylamide gel mass (an elongated Pasteur pipette works well).
7. Add 0.5 times the volume of Acrylamide Gel Elution Buffer to the polyacrylamide precipitate. Spin for a few moments and centrifuge again. Combine the two supernatants.
8. (Optional) Pass the supernatant through a single-use plastic chromatography column (e.g., Isolabs, Inc., Quick-Sep Columns) or a syringe cartridge containing a Whatman GF/C filter membrane or silica-filled glass wool to remove residual polyacrylamide gel.
9. Add 2-fold volume of ethanol at 4°C to the passage solution and place on ice for 30 min. Centrifuge in a benchtop centrifuge at 4°C for 10 min at maximum speed to recover DNA.
10. Dissolve the DNA with 200 μl of TE (pH 8.0), add 25 μl of 3 mol/L sodium acetate solution (pH 5.2), and then precipitate the DNA again with 2 times the volume of ethanol as in step 9.
11. Carefully wash the DNA precipitate with 70% ethanol and redissolve the DNA to a final volume of 10 μl with TE (pH 8.0).
12. Characterize and quantify the DNA by polyacrylamide or high resolution agarose gel electrophoresis.
(1) Mix a small amount of the final prepared DNA fragment (~20 ng) with 10 μl of TE (pH 8.0) and add 2 μl of the desired gel loading buffer.
(2) Sample and electrophoresis on a polyacrylamide or high-resolution agarose gel at the appropriate concentration and use the known endonuclease digestion product as a molecular mass marker. Isolated fragments should migrate in synchronization with fragments of the same size in the molecular mass marker.
(3) Carefully examine the gel for weakly fluorescent bands of DNA contamination. The amount of DNA can often be estimated by comparing the relative fluorescence intensity of the target band with that of the molecular mass marker.