1/15/2024 0 Comments Northern blotting procedure steps![]() ![]() ![]() The third step involves hybridization with probe and washing.During this transfer the RNA fragments retain the same pattern of separation they had on the gel. The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. Once the RNA molecules reach the nylon membrane they become adsorbed tightly to the membrane. This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.įrom the tray now Rises through the gel taking with it the RNA molecules.Again a thick stack of blotting papers or absorbent paper is placed over the membrane.An nylon membrane of the same size as that of the gel is placed over it.Next the gel containing RNA is placed on the top of it.These are placed such that they imbibe the buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. A support for gel on membrane is kept in this buffer. The tray is filled with the suitable transfer buffer. The basis of this transfer is the capillary action. There is the traditional way to transfer the RNA to the Nylon membrane.The separated RNA molecules are now transferred from the gel to the suitable solid support, such as the Nylon membrane. Thus denaturing gel electrophoresis is used in this step. To denature RNA formaldehyde is used as a denaturing agent.Otherwise the secondary structures of RNA molecules will affect their electro phoretic mobility during gel electrophoresis.So if we want to separate them on the basis of their molecular weights we need them to bring in the linear shape. Because of intramolecular base pairing they form secondary structures. This is because RNA molecules fold onto themselves.RNA is a single-stranded nucleic acid but, still this RNA gel electrophoresis also includes the denaturation step.RNA molecules are negatively charged, so they move from negative to the positive electrode during gel electrophoresis.The RNA molecules isolated from cells are separated according to size by Gel electrophoresis. ![]() The first step in northern blotting is RNA gel electrophoresis.Steps of Northern Blotting 1) RNA gel Electrophoresis Northern Blotting is a technique which will be suitable for a study gene expression and analysis. the overall technique and the steps involved in northern blotting is almost similar to the southern blotting. Northern Blotting is a blotting technique for the detection of RNA. ![]()
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