![]() 3) Drain blot and add ECL detection solution. 2) Wash 2 x 5 minutes with 20x SSC at room temperature. Discard solution and wash 2 x 10 minutes at 42C. Day 4-Processing blot 1) Wash blot for 20 minutes in primary wash buffer at 42degreesC. 12) Prepare primary wash buffer and put in hybridization oven to warm to 42 degrees C. 11) Withdraw a small amount of prehybe fluid from blot, mix with probe and add back to blot. 9) Incubate for 10 minutes at 37degreesC (incubate 20 minutes if probe is <300 bp). Mix thoroughly and spin briefly to collect liquid at the bottom of the tube. 7) Add 25 ul of DNA labelling reagent to cooled DNA. 6) Immediately cool on ice for 5 minutes. 5) Dilute DNA to 10 ng/ul (total volume=25 ul) and boil in vigorous boiling water bath for 5 minutes. 4) While prehybing, prepare 10 ng of probe per ml hybridization fluid (250 ng). Mark lanes on Hybond+ with pencil 2) Rinse blot with 6x SSC and bake 2 hrs at 80degrees C. Day 3-Taking down blot and Making the Probe 1) Carefully remove blotting paper. Place in hybridization oven at 42degreesC. Typically prepare 25 ml solution (0.73 g NaCl, 1.25 g blocking mix in 25 ml hybridization solution). Agitate for 30 minutes 8) Set up blot (20x SSC in resevoir and 10x SSC in wicking papers) 9) Prepare prehyb solution (0.5 M NaCl, 5% ECL blocking mix in ECL hybridization solution). 7) Cover gel with neutralization solution. Stop 25 minutes after bromophenol blue has returned to its blue color. 4) Rinse gel with distilled water 5) Cover gel with denaturation solution and agitate. Treat until blue dye turns yellow (approximately 10-12 minutes). 3) Transfer gel to dish, cover with depurination solution and agitate. Note:- The Southern blot method is named after the British Biologist Edwin Southern, who first published it in 1975.Southern Blotting Day 1-Genomic Digests Digest 10 ul of genomic DNA overnight Day 2-Setting up Blot 1) Run gel slowly 2) Photograph gel with ruler (this will allow you to line up the markers with your bands. Used to measure molecular weight of restriction fragments and to determine the relative amounts in different samples. Detection of cancer and prenatal diagnosis of genetic disease So the correct answer is option (A).Īdditional Information: It is also used for -ģ. The Southern Blot technique is for detection DNA. Close relations of an intending immigrant.Ħ. Thus the analysis of the DNA of interest is possible from the required sample.ĥ. Therefore DNA extracted from a single cell is enough to perform DNA fingerprinting analysis. The sensitivity of the technique can be increased by use of polymerase chain reaction (PCR). These bands have a specific characteristic pattern for each individual DNA. Hybridisation is done with a VNTR probe, the autoradiogram gives many bands of different sizes. Detection of hybridized DNA fragments by autoradiographyħ. Hybridisation using labelled VNTR probeĦ. DNA fragments are transferred or blotted to synthetic membranes such as nitrocellulose or nylonĥ. Separation of DNA fragments is done by electrophoresis, where agarose gel is the medium or (RFLP Restriction Fragment Length Polymorphism).Ĥ. This technique, as used earlier, involved Southern blot hybridization using radiolabeled VNTR as a probe. This technique of DNA was developed by Alec Jeffreys. It is also used in immunogenetics and molecular biology.Ĭomplete step-by-step solution:- The basis of DNA fingerprinting is VNTR (a satellite DNA as a probe that shows a very high degree of polymorphism). Hint:-Southern blot hybridization using radioactive VNTR (a satellite DNA as a probe that shows very high degree of polymorphism) is a technique used in DNA fingerprinting which is developed by Alec Jeffreys.
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