Southern blotting (capillary transfer of DNA to a membrane)
Southern blotting (capillary transfer of DNA to a membrane)
Genomic DNA samples are prepared by digestion with one or more restriction endonucleases, and the digested fragments are separated by electrophoresis on a standard agarose gel according to size. the DNA is denatured in situ and transferred from the gel to a solid-phase support (usually nylon or nitrocellulose membranes), where the DNA fragments are retained in place as they are transferred to the membrane. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Southern blotting (capillary method of transferring DNA to a membrane)
Principle
Genomic DNA samples are prepared by digestion with one or more restriction endonucleases, and the digested fragments are separated by electrophoresis on a standard agarose gel according to size. the DNA is denatured in situ and transferred from the gel to a solid-phase support (usually nylon or nitrocellulose membranes), where the DNA fragments are retained in place as they are transferred to the membrane.
Materials and Instruments
Appropriate restriction endonuclease Agarose gel without ethidium bromide Genomic DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Basic transfer buffer Denaturing solution Neutralizing buffer Neutral transfer buffer
Agarose gel without ethidium bromide Cross-linking equipment Glass baking trays Large-diameter yellow pipette tips Neoprene plugs Nylon or nitrocellulose membranes Resin glass sheets or disks Rotary oscillation platforms Thick blotting paper Fluorescently labeled transparent rulers
1. Buffers and solutions
Alkaline transfer buffer (applied to alkaline transfer of nylon membrane) (0.4 mol/L NaOH, 1 mol/L NaCl)
Denaturing solution (applied to neutral transfer) (1.5 mol/L NaCl, 0.5 mol/L NaOH, HCl ( 0.2 mol/L), applied to depurination of DNA)
Neutralizing buffer I ( applied to the transfer of uncharged nylon membrane) (1 mol/L Tris ( pH 7.4), 1.5 mol/L NaCl)
Neutralization buffer Ⅱ (applied to the alkaline transfer of nylon membrane) (0.5 mol/L Tris-Cl ( pH 7.2), 1 mol/L NaCl)
Neutralization Transfer Buffer (10X SSC or 10X SSPE) (6X SSC, 6X Sucrose Gel Sampling Buffer, SYBR Gold or Ethidium Bromide, TE ( pH 8.0))
2. enzymes and buffers
Appropriate restriction endonucleases
3. gels
Agarose gel without ethidium bromide prepared with 0.5X TBE or 1X TAE (0.7%)
4. Nucleic acids and oligonucleotides
DNA Size Markers
Genomic DNA
5. Specialized equipment
Cross-linking equipment (e.g. Stratalinker, Stratagene; GS Gene Linker, Bio-Rad), or microwave oven, or vacuum oven
Glass baking dish
Large-diameter yellow pipette tips
Neoprene stopper
Nylon or nitrocellulose membrane
Plexiglas sheet or glass disk
Rotary oscillation platform
Thick absorbent paper (e.g., Whatman 3 MM, Schleicher&Schuell GB004, or Sigma QuickDraw)
Transparent ruler with fluorescent markers
Heavy weight (400 g )
II. METHODS
1. DNA digestion and electrophoresis
(1) Digest an appropriate amount of DNA with one or more types of restriction endonucleases.
Use a large-diameter yellow pipette tip to manipulate large molecules of DNA.
(2) If desired, concentrate the DNA fragments by ethanol precipitation after digestion. Dissolve the DNA in approximately 25 μl TE (pH 8.0).
(3) Determine the concentration of the DNA digestion product by fluorescence measurement or by ethidium bromide or SYBR Gold spotting assay. Transfer the appropriate amount of digestion product to a new microcentrifuge tube. Add 0.15 times the volume of Sucrose Sampling Buffer and separate the DNA fragments by agarose gel electrophoresis (for most genomic DNA, a 0.7% gel prepared with 1X TAE or 0.5X TBE can be used). Apply a low voltage to the gel (~ < 1 V/cm) to allow the DNA to migrate at a slower rate.
(4) After sufficient electrophoresis, the gel is stained with ethidium bromide or SYBR Gold and photographed. A transparent fluorescent ruler is placed on the side of the gel so that the distance traveled by each DNA band can be read directly from the photograph.
(5) To denature the DNA, transfer the DNA from the gel to a nitrocellulose membrane or a neutral or charged nylon membrane using one of the following methods.
2. Prepare the gel for transfer
(1) After electrophoretic separation of the DNA, transfer the gel to a glass baking dish. Use a sharp razor blade to trim away any unwanted portions of the edge of the gel, including the gel above the spiking wells. Leave enough spiking holes in the gel to mark the location of the spiking holes on the membrane after the DNA transfer is complete. Cut a small triangle in the lower left corner of the gel (with the spiking hole end down) to mark the orientation of the gel during the following procedure.
(2) Denature the gel in denaturing solution (alkaline) for DNA denaturation as follows.
① Transfer to an uncharged membrane
A. Place the gel in 10 times the gel volume of denaturing solution for 45 min at room temperature and gently shake (e.g., on a shaking platform).
B. Briefly soak the gel in deionized water, then immerse the gel in 10 times the volume of the gel in Neutralization Buffer I for 30 min at room temperature and gently shake. Change the neutralization buffer once and continue to soak the gel for 15 min.
② Transfer to a charged nylon membrane.
A. Soak the gel in several times the volume of Basic Buffer for 15 min at room temperature and gently shake (e.g., on a shaking platform).
B. Continue to soak the gel for 20 min with one change of solution and gentle shaking.
3. Prepare the membrane for transfer
(1) Using a clean scalpel or paper cutter, cut a piece of nylon or nitrocellulose membrane 1 mm larger than the gel on each side. Cut two sheets of heavy absorbent paper the same size as the membrane.
(2) Float the membrane in a dish of deionized water until the membrane is completely wet from the bottom up, then immerse the membrane in appropriate transfer buffer for at least 5 min. use a clean scalpel blade to cut off a corner of the membrane to match the corner cut off by the gel.
4. Assembly of the transfer device and transfer of DNA
Neutral transfer buffer (10X SSC or 10X SSPE) is required for transferring DNA to uncharged membranes. Basic transfer buffers 0.4 mol/L NaOH and 1 mol/L NaCl should be used for transferring DNA to charged nylon membranes.
(1) During denaturation of DNA, a piece of thick blotting paper is placed on a piece of Plexiglas plate or glass dish to form a support longer and wider than the gel. The ends of the blotting paper need to hang down from the edge of the dish. Place the support in a large dry baking dish. The support can be placed on four neoprene plugs to elevate it from the bottom of the dish.
(2) Place an appropriate transfer buffer in the dish until the level is almost flush with the surface of the support. When the blotting paper on the support is completely moistened, a glass rod or pipette is used to drive out air bubbles.
(3) Remove the gel from the solution and invert it so that the original bottom surface is up. Place the inverted gel on the support and in the center of the blotting paper.
(4) Surround the gel with Saran wrap or Parafilm, but do not cover the gel.
It is easy to hang down from the edge of the gel and make contact with the platform. This short-circuiting is a major cause of inefficient DNA transfer from the gel to the membrane.
(5) Wet the gel with an appropriate transfer buffer. Place the moistened membrane on the gel and overlap the tangents. To avoid air bubbles, one corner of the membrane should be in contact with the gel before slowly placing the membrane on the gel. One edge of the membrane should be just beyond the edge of the line of sample holes at the top of the gel.
IMPORTANT: Do not move the membrane once it has been placed on the gel surface. There should be no air bubbles between the membrane and the gel.
(6) Moisten two sheets of thick blotting paper with appropriate transfer buffer and place on the moistened membrane. Use a pipette to drive out trapped air bubbles.
(7) Cut or fold a stack of paper towels slightly smaller than the blotting paper (5 to 8 cm high). Place the paper towel on top of the blotting paper. Place a glass plate on top of the paper towel and compact it with a 400 g weight.
(8) DNA transfer should be carried out for 8~24 h. Replace the tissue with a new one when it becomes wet. Try to avoid wetting the entire stack of paper towels with buffer.
(9) Remove the paper towel and blotting paper from the gel. Turn the gel and the membrane in contact with it over and lay the gel flat on dry blotting paper, gel up. Mark the location of the spiking hole with a very soft pencil or ballpoint pen.
(10) Peel the gel from the membrane and discard the gel.
5. Fixing DNA on the membrane
The sequence of steps from immobilization of DNA on the membrane to subsequent hybridization depends on the type of membrane, the method of transfer, and the method of immobilization. It is not necessary to immobilize the DNA on the membrane prior to hybridization because the basic buffer will cause the DNA to covalently bind to the positively charged nylon membrane. DNA transferred to uncharged nylon membranes in neutral buffer needs to be vacuum baked or microwaved to immobilize the membrane, or crosslinked to the membrane by UV irradiation. 
(1) Immerse the membrane in an appropriate amount of one of the following solutions:
Neutral transfer: 6X SSC, 5 min at room temperature.
Alkaline transfer: Neutralization buffer II [0.5 mol/L Tris-Cl (pH 7.2) and 1 mol/L NaCl], room temperature for 15 min.
(2) Fixation of DNA transferred to uncharged membrane
① Fixation by vacuum oven
A. Remove the membrane from the 6X SSC and allow excess liquid to drain off. Place the membrane on a piece of paper and let it dry at room temperature for 30 min.
B. Sandwich the membrane between two sheets of dry blotting paper. Bake in a vacuum oven at 80°C for 30 min ~ 2 h.
② Fix by baking in microwave oven
A. Place the moist membrane on a piece of dry absorbent paper.
B. Turn the microwave oven to maximum power (750~900 W) and heat the film for 2~3 min.
③ Cross-linking by UV irradiation
A. Place the moist membrane on a piece of dry absorbent paper.
B. 254 nm irradiation crosslinks the DNA to the membrane (Khandjian 1987).
(3) Hybridize the immobilized DNA directly with the probe.