518 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS
Southern blotting
Ed Southern
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Correspondence should be addresd to E.S. (ed.ac.uk).Published online 27 June 2006; doi:10.1038/nprot.2006.73
This protocol describes a basic method to perform the Southern blot. Blotting allows the detection of specific molecules among a mixture parated by gel electrophoresis. Molecules are transferred from the gel to a porous membrane by capillary action using absorbent paper to soak solution through the gel and the membrane. For DNA, specific quences are detected in the membrane by molecular hybridization with labeled nucleic acid probes. The original method, on which this protocol is bad, ud labeled RNAs to detect specific DNA fragments in genomic DNA that had been digested with restriction endonucleas. This protocol can be completed in 1–5 d and is inexpensive to carry out, as it requires only basic laboratory equipment.
INTRODUCTION
Blotting methods are an adjunct to gel electrophoresis, a method for parating DNA 1, RNA 2 and proteins 3 with exceptional resolv-ing power. They allow the detection of specific molecules among the mixture parated in the gel. All methods have in common a step in which molecules are transferred from the gel to a porous membrane, most often achieved by soaking solution through the gel and the membrane using absorbent paper. More complex equipment is available from a number of suppliers for ‘electroblot-ting’4; this is particularly uful for transfer from polyacrylamide gels, which are less porous than agaro. For DNA and RNA 5, spe-cific quences are detected in the membrane by molecular hybrid-ization with labeled nucleic acid probes; proteins are detected using labeled antibodies. The original protocol 6 was adapted for radioactive probes labeled with, for example, 32P , 3H, 35S or 125I. Since then, other labeling systems have been developed, includ-ing fluorescent and chemiluminescent reagents. There have been other modifications to the original method: DNA probes are now commonly ud 7,8, rather than RNA; nylon membranes have replaced nitrocellulo for many applications; transfer is carried out in alkaline rather than neutral solution to avoid reassociation of DNA quences during transfer; DNA is reduced in size by acid treatment to improve transfer of large fragments; and the proce-dure has been adapted to gel formats differing from the gel strips and tube gels ud in the original protocol. But modern protocols retain many of the features of the original.
The method was ud in veral key studies. The original pro-posal for the genetic mapping of the human genome 9 was bad on restriction fragment length polymorphisms (RFLPs) detected by blotting 10. Introns were first en in blots of rabbit genomic DNA hybridized with probes for the β-globin gene 11. The first ‘DNA fingerprints’ were produced by hybridizing restriction digests of human DNA with minisatellite probes 12. Many of the early applications of the method have been replaced by Sanger quencing, which gives more information, or methods bad on the polymera chain reaction (PCR), which is simpler to perform. Blotting is still ud in a number of applications, such as measuring copy number of transgenes or analyzing stretches of DNA too long to be amplified by the PCR or quenced using Sanger’s method. It is also ud in studies of DNA structure in which different physical forms are parated by two-dimensional gel electrophoresis and specific components detected by blotting 13.
The whole process involves a number of steps: purification of the DNA to be analyzed, digestion with restriction enzyme, gel electrophoresis, transfer from gel to membrane by blotting, prepa-ration and labeling of probe, and detection of label. Most, but not all, of the steps are described here. I prent the basic ini-tial protocol and a few of the various modifications that have been made over the years. Several methods have been developed for the transfer tup; tho prented here are b
ad on the original, with minor modifications. I hope that the variations and others will be discusd in the comments attached to this protocol by current urs of the method.
MATERIALS
REAGENTS
• Electrophoresis buffer (TAE or TBE; e REAGENT SETUP)• Electrophoresis-grade agaro • E thidium bromide (0.5 µg ml –1, dissolved in H 2O) or other DNA staining agent (there are veral alternatives for staining DNA—for example, SYBR Green—that are claimed to be safer than ethidium bromide)
! CAUTION Ethidium bromide is mutagenic and so needs to be handled with care. Wear gloves at all times and dispo of pipette tips according to the relevant safety regulations. Be careful not to touch anything with gloves contaminated with ethidium bromide.
• 2× and 20× SSC (20× SSC is 3.0 M NaCl, 0.3 M sodium citrate)• 6× loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol in water
• DNA molecular weight markers • P rehybridization and hybridization mix: 6× SSC, 0.5% SDS, 5× D
enhardt's solution and 100 mg ml –1 denatured, sheared salmon sperm DNA or yeast tRNA • D enhardt’s solution: 0.02% polyvinylpyrrolidone, 0.02% Ficoll and 0.02% bovine rum albumin (BSA)
• Paraffin oil (required only for Step 20A)• C ellulo nitrate or nitrocellulo filter (e.g., Millipore 25 HAWP; nylon
membranes for blotting are obtainable from veral suppliers under different trade names)
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NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 519
• RNa A (20 pg ml –1 in 2× SSC)• Restriction enzyme and buffer • R adioactively labeled RNA ! CAUTION When dealing with radioactivity, appropriate safety precautions must be followed and appropriate national regulations adhered to.• 2,5-Diphenyloxazole (PPO) in toluene (20% wt/vol; required only for detecting RNA labeled with 3H, 35S, 125I or
14C)
EQUIPMENT • F or transfer from narrow strips of gel (Fig. 1
): three pieces of glass or Perspex (also known as Plexiglas), 5 cm × 20 cm and the same thickness as the gel
• Four or five pieces of thick, dry filter paper or paper towels, 10 cm ×
18 cm
• H ybridization vesl (required only for Step 20B) made from Perspex with
internal dimensions of 0.8 mm deep × 2 cm high × ~1 cm longer than the membrane to be hybridized (note that there are various alternative
methods of hybridization; tho prented here, which are from the original publication, do not allow for mixing)• For transfer from wide gels (Fig. 2):• F our narrow pieces of Perspex the same thickness as the gel and long enough to surround it with a space of ~3 mm
• A tray ~20–50 mm deep and ~20 mm longer and wider than the gel • A glass sheet long enough to sit on the tray and narrow enough to leave a gap ~10 mm on each side
• S everal pieces of thick filter paper larger in area than the gel, long enough to cover the glass sheet and dip into the tray • A piece of nitrocellulo membrane, moistened with 2× SSC, large enough to cover the gel and rest on the four strips of Perspex strips • A stack of paper towels • Gel casting apparatus
• Gel tank for electrophoresis • Power supply REAGENT SETUP
DNA The following procedure should initially be carried out using a range of DNA concentrations digested with a panel of different enzymes to
determine the optimum DNA concentration and restriction enzyme to u. In general, up to 1 µg of clonally derived DNA (for example, from plasmid or bacteriophage clones) is more than sufficient, even for low-copy-number plasmids. Larger amounts will be required when more complex DNA (for example, genomic DNA) is to be parated. A good starting range would be 5–10 µg.保护视力小常识
Electrophoresis buffer U TAE (40 mM Tris, 20 mM acetic acid, 1 mM
EDTA, pH 7.4–8.2, normally made as 20× stock) or TBE (89 mM Tris, 2.5 mM EDTA, 89 mM borate, normally made as a 10 × stock). TAE is generally best when gels are to be run for a short period of ti
me and when DNA fragments are to be recovered from the gel. TBE is a better buffer, and should be ud when gels are to be run for more than 2 h.
PROCEDURE
Restriction digestion of DNA ● TIMING 2–24 h
1| Add the components detailed in Table 1avoiding bubbles.
▲ CRITICAL STEP The enzyme should always be added last, and should be kept at −2| 3| It may be necessary to concentrate the DNA after digestion to ensure it is in a suitable volume to be easily e Step 10 below. Standard ethanol precipitation followed by resuspension in ddH 2O can usually be ud to concentrate DNA samples. Be careful to remove traces of ethanol, which could cau the sample to float out of the wells during gel loading.
Figure 1 | Steps in the procedure for transferring DNA from an agaro gel strip to a hybridization membrane. (a ) Place a glass or Perspex sheet on filter paper soaked in 20× SSC. Add the gel parallel to the glass or Perspex and 2–3 mm away from it. Lay another piece of glass or Perspex 2–3 mm away on the other side of the gel. (b ) Place the nitrocellulo membrane on top of the gel with it
s edges resting on the Perspex or glass so that it bridges the air space. (c ) Place two moist filter paper strips on top, aligned so they are directly above the air spaces and the Perspex or glass. (d ) Place dry filter paper across the whole transfer tup.
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520 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS
Electrophoresis ● TIMING 1–16 h
如何选专业4| Decide on the percentage and size of gel required. The percentage
depends on the size of the fragments to be parated; a guide is given in
Table 2. The size of the gel required depends on the range of fragments to be parated—generally, longer gels will be
needed to ensure sufficient paration
of genomic DNA and of multiple
fragments of similar size. A 0.7–2% gel
is sufficient for most applications. To
obtain sufficient paration of fragments
for some genomic samples, it may be necessary to run a low-percentage gel.
For gels under 0.8%, you may need to
u a high-percentage (for example, 2%) ba gel underneath, as low-percentage
gels are very fragile. Remember to pour the ba gel without the comb in place.
Once it has t, pour the low-percentage gel on top with the comb in place, ensuring that the bottom of the comb does not touch the ba gel.
5| Make up 1× electrophoresis buffer by diluting the appropriate amount of stock solution in ddH 2O. Add the correct amount of agaro to the 1× buffer in a conical flask, swirling to ensure that no clumps are formed. For example, for 150 ml of a 1.5% agaro gel, dissolve 2.25 g agaro in 150 ml electrophoresis buffer. 150 ml is sufficient volume for one 18 cm × 20 cm gel. Melt the
agaro either in a microwave or on a heated stirrer, t at a high heat and a gentle stirring speed. When using a microwave, the gel should be swirled gently every 30 s or so to ensure the gel melts evenly. The gel should be completely melted within 3 min on the default tting of most standard microwaves.
菊花的形态! CAUTION Check the agaro often and never leave it unattended when heating, as it is liable to boil over very quickly. To minimize this risk, always try to u a conical flask that is three or four times larger than the volume of agaro required. Be careful of superheating, which may cau the gel to suddenly boil over when the flask is moved.
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6| Transfer the flask containing the molten gel to a magnetic stirrer. Allow the gel to cool slowly to around 50–60 °C, while stirring gently. If ethidium bromide is to be included in the gel, it should be added at this stage to 0.5 µg ml –1.
7| While the gel is cooling, prepare sufficient 1× electrophoresis buffer (with ethidium bromide to 0.5 µg ml –1, if included in the running buffer) to fill the rervoir of the gel tank. Pour the buffer into the tank so that it is a few millimeters above the level of the gel support.
8| Prepare the gel cast, choosing a comb with teeth sized to form wells that will comfortably hold the sample volume after the addition of loading buffer. Ensure that the gel cast (with the comb in place) is on a level surface and slowly pour the gel.
▲ CRITICAL STEP Try to avoid letting bubbles form, as they will cau the DNA to run irregularly. Bubbles can usually be removed by bursting them with a yellow pipette tip. This should be done as soon as the gel has been poured.9| Once the gel has t (at which point it should be opalescent in appearance), gently remove the comb. Transfer the gel to the gel tank and add more 1× running buffer to cover the gel. Add as much buffer as possible without exceeding the limit of the tank—this is particularly important if the gel is to be run for a long time, as it maximizes buffering capacity and minimizes the chance of the gel melting due to incread heat.
× SSC
× SSC Figure 2| Transfer from wide gels. (a ) Stack of paper towels weighted with a glass plate. (b ) Cellulo nitrate or nylon membrane. (c ) Gel surrounded by plastic strips that support the edges of the membrane
and the paper towels. (d ) Wad of filter paper to act as a wick to transfer the 20× SSC from the tray to
the gel. (e ) Tray of 20× SSC with a glass plate to support the filter paper wick.
Table 1 | Components of a typical restriction digest of genomic DNA Reagent Amount
DNA (1 µg µl −1)
岳阳楼记通假字10 µl (10 µg)10× buffer (as supplied with the enzyme)10 µl (1× final concentration)ddH 2O 75 µl
Enzyme (10 U µl −1)
5 µl (50 U)a Total 100 µl
a For clonally derived DNA, 1 µl of enzyme in a total digest volume of 20 µl is usually
sufficient.
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NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 521
▲ CRITICAL STEP It is important to be gentle at this point so as not to tear the wells. If the gel apparatus allows, the comb can be removed after the gel has
the gel before removing the comb.
10| Prepare the samples by adding
loading buffer to a final concentration of
1× (for a 6
× stock, add 1 µl buffer for every 5 µl of sample). Stir gently with the pipette tip to mix, avoiding air bubbles, which will cau the sample to float out of the wells.
11| Carefully pipette the samples into the wells. It is important to also include at least one well (preferably two) of appropriate molecular weight markers, which usually come prepared with loading buffer. This allows the size of fragments in the samples to be determined.
▲ CRITICAL STEP Place the pipette tip just below the top corner of the well—do not push the pipette tip right to the bottom, as this can puncture the well or cau the sample to spurt out of the well. Releasing the sample slowly will also minimize the risk of the sample floating out of the well.
12| Carefully attach the lid to the gel tank, and attach the lid to the power supply. Set the voltage app
ropriately and switch on the power supply. The progress of the paration of DNA fragments can be followed by the progress of the dye fronts. Small bubbles ▲ CRITICAL STEP DNA is negatively charged and will therefore run toward the positive electrode. Ensure that the lead clost to the wells is attached to the negative socket of the power supply (usually black) and the lead furthest from the wells is attached to the positive socket (usually red).
Preparation for transfer ● TIMING 2–3 h
13| If the gel was run in the abnce of ethidium bromide, immer the gel for 0.5–2 h in electrophoresis buffer containing ethidium bromide (0.5 µg ml –1).
14| Transfer the gel carefully to a transilluminator rigged to a camera. Photograph the gel in ultraviolet light (256 nm; high concentrations of DNA can also be detected at 302 or 356 nm) with a red filter on the camera. Lay a ruler alongside the gel (this aids in matching the photograph of the fluorescence of the DNA to the final radioautograph of the hybrids). If the DNA has not parated sufficiently, the gel can be returned to the tank and run for longer. Clonal DNA should appear as distinct bands. Genomic DNA should appear as a smear, with brighter bands that reprent repetitive DNA elements. At this point, the area of gel to be 16| Replace the 1.5 M NaCl, 0.5 M NaO
H solution with 3 M NaCl, 0.5 M Tris HCl (pH 7) and leave for a further 15–30 min. This step neutralizes the gel.
Transfer tup ● TIMING ~15 min
17| For the transfer of narrow strips (illustrated in Fig. 1), prepare the components for the gel transfer so that there is one piece of thick filter paper (20 cm × 18 cm) soaked in 20× SSC and one piece of nitrocellulo filter (of equal length to the gel strip and approximately 1 cm wider) soaked in 2× SSC. For transfer from larger areas of gel, asmble the components illustrated in Figure 2.▲ CRITICAL STEP Immer the filter paper and transfer membrane by first floating them on the surface of the solution; otherwi air will be trapped in patches, which leads to uneven transfer.
Table 2 | Separation of linear DNA fragments in agaro gels
Agaro in gel (percent)Efficient range of paration of linear DNA (kb)
0.360–5.00.620–1.00.710–0.80.97–0.51.26–0.41.53–0.22.02–0.1
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522 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS
18| For narrow strips, lay the large filter paper soaked in 20× SSC on a glass or plastic surface, taking care to avoid trapping air bubbles below the paper. Pour 20× SSC on top so that the surface is glistening wet. For wider gels, fill the tray with 20× SSC and lay a glass plate over it; soak a wad of filter paper in 20× SSC and lay it over the plate, dipping into the tray (Fig. 2).19| Lay a glass or Perspex sheet on top and to one side of the wet paper.
20| Remove the gel from the neutralizing solution and lay it parallel to the glass or Perspex sheet, 2–3 mm away from it.21| Lay the cond glass or Perspex sheet 2–3 mm away from the other side of the gel.
22| Lay the nitrocellulo membrane on top of the gel with its edges resting on the sheets of Perspex or glass, so that it bridges the two air spaces. Take care in aligning the gel before it makes contact with the gel and lay it down by ‘rolling’ from one edge to avoid trapping air between the gel and the membrane.
▲ CRITICAL STEP Once it is placed, do not move the membrane, as transfer of DNA may already have taken place.
23| Place the absorbent paper on top.
▲ CRITICAL STEP Do not put a heavy weight on top, as this may flatten and distort the gel during the transfer process, making final size comparisons more difficult.
Transfer ● TIMING 3–16 h for transfer and 1–2 h for post-transfer processing
24| Let the transfer tup stand to allow transfer to take place. The minimum time required for complete transfer depends on the size of the fragments and the gel concentration. For example, a period of 3 h is enough to transfer completely all Hae III fragments of E. coli DNA from 2% agaro gels 3 mm thick. But even after 20 h, transfer of large Eco RI fragments of mou DNA from 9-mm-thick gels is not complete.
▲ CRITICAL STEP During transfer it may be necessary to occasionally add more 20× SSC to the bottom sheet of filter paper. If the paper dries too much, the gel will shrink against the nitrocellulo membrane and liquid contact will be broken. The paper may be flooded, but care must be taken that liquid does not fill the air spaces between the gel and the side pieces and soak the paper, bypassing the gel. It may be convenient to leave the nitrocellulo in position overnight: if the supply of 20× SSC has dried up, it will be found that the gel has shrunk against the nitrocellulo, but this will not i
mpair the transfer.
25| At the end of the transfer period, lift the nitrocellulo membrane carefully so that the gel remains attached to its underside.26| Turn over the nitrocellulo membrane and mark the outline of the gel in pencil by making a ries of dots.
27| Peel the gel off the nitrocellulo. Any DNA remaining in the gel can be en by viewing the gel in ultraviolet light
(256 nm) with a red filter on the camera, as the ethidium bromide is not completely removed during treatment of the gel and will still fluoresce.
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28| Cut the nitrocellulo with a flamed blade so that just the area that was in contact with the gel remains (described from this point onward as the membrane).
29| Immer the membrane in 2× SSC for 10–20 min.
30| Bake the membrane in a vacuum oven at 80 °C for 2 h. Alternatively, the DNA may be cross-linked to the membrane by exposure to short-wavelength ultraviolet light; commercial equipment is available for this.
■ PAUSE POINT The membranes can be stored at room temperature for prolonged periods. It is probably wi to keep them dry.Blocking ● TIMING 1 h
31| Block the membrane by incubating it in Denhardt’s solution for 1 h or more.
Hybridization ● TIMING 1–16 h depending on probe complexity and concentration
32| Radioactive RNAs from cells are usually available only in small quantities, and for the original protocol it was important to keep the volume of the solution ud for hybridization as small as possible to ensure a reasonable concentration of RNA. Two procedures (A and B) were ud for hybridizing the nitrocellulo membranes after transferring the restriction
陈国公主fragments. Procedure A us the smaller volume. Procedure B us a vesl designed to hold the membrane in a small volume
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