What are the different bands of DNA?
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Agarose gel electrophoresis
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be purified from the gel if necessary.The agarose gel is made by dissolving the solid agarose powder in the electrophoresis buffer. Usually Tris-AceticAcid-EDTA ("TAE") buffer is used. A commonly used stock solution for TAE is 50 times concentrated ("50xTAE"; the standard procedure for preparation of 50xTAE is: mix deionised water with solid Tris powder, a certain amount of an EDTA stock solution, usually 0.5M EDTA pH8.0, and concentrated acetic acid to adjust the pH to 7.6). The agarose will only fully dissolve when boiled for a few minutes and the warm gel solution then is poured into a mold which is fitted with a well-forming comb. Cooling down to room temperature results in the slow formation of a solid gel when the concentration of agarose is between 0.5 and 2% (weight/volume).To make the DNA visible in the gel, ethidium bromide is added to the gel solution and the buffer (it can also be left out of the gel and buffer; staining of the gel can be done in that case after the gel run..). This positively charged polycyclic aromatic compound binds to DNA by inserting itself between the basepairs ("intercalation"). The DNA bands can be seen by exposure of the gel to ultraviolet light, due to the the large increase in fluorescence of the ethidium bromide upon binding to the DNA.Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. This buffer both conducts electric current and controls the pH of the solution during electrophoresis. DNA samples for loading into the wells ("slots") of the gel are prepared by addition of a tracking dye (e.g. Orange G or Bromo Phenol Blue)which also contains a component (usually glycerol or sucrose) to increase the density of the sample to facilitate the loading.Electrophoresis usually is at about 5 Volts per cm for 0.5 - 2 hours or more at room temperature, depending on the desired separation. Size markers may be co-electrophoresed with DNA samples, when appropriate for fragment size determination. Many commercial size-marker sets are available with different size ranges.After electrophoresis, the gel is placed on a UV light box and a standard or digital photograph of the fluorescent ethidium bromide-stained DNA separation pattern is taken.
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Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be purified from the gel if necessary.The agarose gel is made by dissolving the solid agarose powder in the electrophoresis buffer. Usually Tris-AceticAcid-EDTA ("TAE") buffer is used. A commonly used stock solution for TAE is 50 times concentrated ("50xTAE"; the standard procedure for preparation of 50xTAE is: mix deionised water with solid Tris powder, a certain amount of an EDTA stock solution, usually 0.5M EDTA pH8.0, and concentrated acetic acid to adjust the pH to 7.6). The agarose will only fully dissolve when boiled for a few minutes and the warm gel solution then is poured into a mold which is fitted with a well-forming comb. Cooling down to room temperature results in the slow formation of a solid gel when the concentration of agarose is between 0.5 and 2% (weight/volume).To make the DNA visible in the gel, ethidium bromide is added to the gel solution and the buffer (it can also be left out of the gel and buffer; staining of the gel can be done in that case after the gel run..). This positively charged polycyclic aromatic compound binds to DNA by inserting itself between the basepairs ("intercalation"). The DNA bands can be seen by exposure of the gel to ultraviolet light, due to the the large increase in fluorescence of the ethidium bromide upon binding to the DNA.Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. This buffer both conducts electric current and controls the pH of the solution during electrophoresis. DNA samples for loading into the wells ("slots") of the gel are prepared by addition of a tracking dye (e.g. Orange G or Bromo Phenol Blue)which also contains a component (usually glycerol or sucrose) to increase the density of the sample to facilitate the loading.Electrophoresis usually is at about 5 Volts per cm for 0.5 - 2 hours or more at room temperature, depending on the desired separation. Size markers may be co-electrophoresed with DNA samples, when appropriate for fragment size determination. Many commercial size-marker sets are available with different size ranges.After electrophoresis, the gel is placed on a UV light box and a standard or digital photograph of the fluorescent ethidium bromide-stained DNA separation pattern is taken.
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