Rinse the membrane in 2× saline sodium citrate (SSC), then place it on a sheet of Whatman 3MM paper and let it dry thoroughly.Mark the positions of the wells on the membrane and cut one corner to mark the orientation. Place a glass plate on top of the stack and put a 0.2 to 0.4-kg weight on the top to hold the stack in place.As each layer is applied, wet it with 20× saline sodium citrate (SCC) and remove air bubbles if any by carefully rolling a 10-ml glass pipet over the surface. Assemble the transfer stack consisting of a sponge, Whatman 3MM paper, the gel, pieces of plastic wrap, a nylon membrane equilibrated in distilled water or nitrocellulose membrane, five pieces of Whatman 3MM paper, and a 4-cm stack of paper towels.Treat the gel with distilled water ∼10 gel volumes 0.25 M HCl for 30 minutes, again with distilled water ∼10 volumes denaturation solution (1.5 M NaCl/0.5 M NaOH) twice for 20 minutes, and then with distilled water ∼10 volumes neutralization solution (1.5 M NaCl/0.5 M Tris-Cl, pH 7.0) for 20 minutes for twice, with gentle shaking at room temperature.Digest the DNA using the restriction endonuclease(s), run in an agarose gel with appropriate DNA size markers, dye the nucleotide strands with ethidium bromide, and identify the band position by visualizing the membrane.Southern blotting onto nylon or nitrocellulose membrane ( Brown, 2001) Then, the membrane is placed in a solution of labeled RNA, single-stranded DNA, or oligodeoxynucleotide complementary to the blot transferred DNA bands to be detected. After transfer, the DNA is fixed onto the membrane. Nitrocellulose or nylon serves as the membrane material. The DNA molecules are run on the membrane. The hybridization membrane is sandwiched between the gel and paper towels which draw the transfer buffer through the gel by capillary action. The agarose gel for southern blotting is mounted on a filter paper dipped in a reservoir containing transfer buffer. The desired DNA is detected using a labeled probe complementary to the desired DNA. The nitrocellulose filter is sandwiched between the gel and the stack of paper towels which draws the transfer buffer from the gel through capillary action. The technique involves the transfer of electrophoresed DNA from the electrophoresis gel to a nitrocellulose membrane. The DNA fragments are separated based on their size and charge during electrophoresis. Southern blotting is based on the principle of separation of DNA fragments by gel electrophoresis followed by the identification by labeled probe hybridization. In addition to its use for DNA analysis, the immunologists have long used the southern blotting for gene identification in somatic rearrangements and transgene studies. Furthermore, southern blotting is used to identify methylated sites in particular genes. Southern blotting could be used for homology-based cloning by exploring the amino acid sequence of the protein product of the gene of interest. Sequences hybridizing with the hybridization probe are further analyzed to obtain the full-length sequence of the gene of interest. These oligonucleotides are chemically synthesized, radiolabeled, and then used for the cloned DNA fragments. Oligonucleotides, similar to the target sequence are designed. After immobilization, the DNA fragments could be subjected to hybridization analysis, enabling the bands with sequence similarity to a labeled probe. The method involves the transfer of DNA fragments from an electrophoresis gel to a membrane causing immobilization of the DNA fragments and bands are produced. Southern, who developed the technique in the 1970s. Southern blotting is a hybridization technique used for deoxyribose nucleic acid (DNA). Balances, Scales and Weighing Equipment.
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