Plant Lab Protocols / Methodology for Nucleic Acids
Southern Blotting
By using gel electrophoresis in combination with restriction endonuclease analysis, a detailed and accurate physical map of a DNA molecule can be constructed. However, in order to construct a genetic map indicating the size and location of genetic coding regions on this molecule more information is required. Further experiment on the DNA fragments in agarose gel is difficult as the gel is very delicate to handle. This has been made possible by transferring the DNA fragments by elution or electrophoretically to nitrocellulose. This 'blotting' technique was first described by Southern in 1975 and is called 'Southern blotting'.
By this technique the separated DNA fragments are transferred and immobilized on the nitrocellulose. It can then be hybridized using a radioactively labeled (mRNA/nick-translated DNA) probe to identify the appropriate gene among the fragments. The procedure for blotting is described below.
Principle
The DNA molecules in the agarose gel are transferred to nitrocellulose membrane either by capillary action (capillary blotting) or by electrophoresis (electroblotting). The smaller fragments are more readily transferred than the larger ones. The DNA molecules are immobilized to nitrocellulose when heated at 80°C under vacuum after transfer.
Materials
» Nitrocellulose filters
» Whatman 3 filter papers
» Denaturing solution
NaOH (0.5M) 20g
NaCl (1.5M) 87g
Water to 1L
» Neutralizing solution (pH 7)
Tris-HCl (0.5M) 60.5g
NaCl (3.0M) 174g
Water 1L
» Saline sodium citrate (SSC) pH 7
Sodium citrate (0.3M) 88.2g
Sodium chloride (3M) 174g
Water 1L
Prepare 10-fold concentrated SSC and store.
Procedure
1.
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Transfer the agarose gel, after electrophoresis, to a suitable large dish A thin sheet of plastic, slightly larger than the gel is used for this transfer.
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2.
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Immerse the gel for 15 min in the denaturing solution.
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3.
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Replace with fresh solution and continue soaking for another 15 min.
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4.
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Repeat the procedure twice with neutralizing solution.
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5.
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In a suitable glass tray place a glass plate (somewhat larger than the gel) on a platform at a height of about 2cm.
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6.
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Place on top of three large pieces of Whatman 3MM paper to serve as a wick. Add 10 X SSC to the tray and wet the wicks completely.
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7.
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Over the wick, place 10 sheets cut exactly the same size of the gel to be blotted. Wet these sheets also and press out any air bubbles between the sheets.
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8.
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Place the pre-treated gel on top of this stack between two plastic spacers across the wick.
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9.
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Cut the nitrocellulose filters to the same width but slightly longer than the size of gel, gently float it on 2 X SSC, thoroughly wet and immerse down.
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10.
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Wet the surface of the gel with 2 x SSC. Place the wet nitrocellulose on the gel starting at the middle and extending along both the ends finally the ends lying over the spacers. Express out any air bubbles between the gel and filter carefully using forceps.
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11.
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Place five sheets of Whatman 3MM paper (gel size) above the gel and then a wad of tissue papers. Place a glass or plastic plate over the top and add a weight on it (a brick or a flask with one liter of water).
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12.
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Allow the transfer to proceed overnight or even longer. The buffer moves upward by capillary action via the gel and filter. During this process the DNA fragments are transferred out of the gel and retained on the nitrocellulose because of its greater affinity, thus making a replica of gel pattern.
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13.
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Dismantle the assembly and recover the filter. Soak in 2 x SSC for 15 min. Observe under UV illumination. The ethidium bromide will transfer with the DNA so it should be possible to visualize some pattern.
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14.
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Dry the filter by placing between two layers of 3MM paper and placing in a vacuum oven (3h at 80°C). The DNA will be immobilized by this drying.
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Notes
1.
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Keep the gel and nitrocellulose filter grease-free. Wear disposable gloves and use forceps whenever handling the filter.
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2.
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There are a number of ways by which blotting is set. Make sure in any case that the buffer flows through the gel and filter. The upper layer of papers should not touch the bottom layers.
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3.
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Nitrocellulose filter should not be dried in a hot air oven as the filter may explode.
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4.
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The dried filter may be further analyzed by hybridization (southern hybridization) using a radioactivity labeled DNA as a probe to identify a particular gene.
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5.
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Transfer of RNA from gel to filter is termed as 'Northern blotting' and that of protein as 'Western blotting'. The latter procedure is described elsewhere in the book.
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References
1. Southern, E (1975) J mol Biol 98 503.
2. Southern, E (1979) In: Methods in Enzymol 68 (Ed Wee, G) Academic Press New York p 152.
3. Manual on Techniques in Molecular Biology (Nucleic Acids) (1986) Workshop held at the Department of Biochemistry Tamil Nadu Agricultural University Coimbatore p 35.
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