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1、Southern blot,动物生物技术系,Southern印迹是将DNA片断从电泳凝胶上直接转移至膜支持物（如硝酸纤维素膜、尼龙膜）上，使DNA片断固定的技术。,Southern印迹,先将DNA经限制性内切酶消化成一系列片段， 进行琼脂糖凝胶电泳，各片段因分子量不同而彼此分开， 然后经碱处理凝胶，使DNA的片段被变性、中和并通过毛细作用在高盐缓冲液中在原位将单链核酸转印到硝酸纤维膜上，烘干、固定。,分子生物学中使用的标记方法,杂交研究中的核酸探针,核酸杂交探针使用的标记物,随机引物的探针标记,DNA的限制性酶切,Southern 印记,DNA分子,限制片段,限制性酶切割,琼脂糖电泳,转移至硝
2、酸纤维素膜上,与放射性标记DNA探针杂交,放射自显影,带有DNA片段的凝胶,凝胶,滤膜,用缓冲液转移DNA,吸附有DNA片段的膜,Southern 印迹法,Albert Lasker Award for Clinical Medical Research,Alec John Jeffreys and Edwin M. Southern Alec John Jeffreys and Edwin M. Southern for development of two powerful technologies - Southern hybridization and DNA fingerprinti
3、ng - that together revolutionized human genetics and forensic diagnostics.,Alec John Jeffreys University of Leicester (UK),Edwin Southern University of Oxford (UK),Southern 印迹法的历史背景,Southern Hybridization,An agarose gel is prepared and run according to the standard procedures, then it is stained and
4、 photographed to have a record of all the DNA fragments. The gel is prepared by first denaturing, then neutralizing, the DNA fragments in the gel,Southern hybridization has many steps and usually requires more than one day (or lab period) to complete. This procedure can be broken down into several s
5、ections,Preparing the gel,After neutralization, the gel is carefully measured. Once the gel dimensions are known, we can begin to assemble the materials needed for Southern transfer.,The nylon membrane is cut to the size of the gel. The membrane itself is sandwiched between two sheets of protective
6、paper. However, wear gloves when working with the membrane to further protect it from oil and dirt. Two pieces of 3 mm filter paper are also cut to the size of the gel. Students should also prepare the stack of paper towels that are placed on top of the membrane. These should be roughly the same siz
7、e as the gel,Cut parafilm strips that are longer than the width and length dimensions of the gel. These are used to “mask“ the gel and direct the capillary action through the nylon membrane. Finally, two large 3 mm filter paper wicks are cut for the next step; assembling the buffer tank and wick.,Fi
8、rst, pour buffer into the large pan. Then, carefully wet the wicks. Once the 3 mm filter paper is wet it can tear easily. Next, invert the small pan and place into the larger pan. Finally, carefully center the wicks over the small pan. Make sure there is enough wick on each side to reach into the bu
9、ffer well.,Assembling the buffer tank and wick,A wicking system is assembled that will draw 10x SSC buffer from a well in a large pan up to the gel. The gel will sit on the wicks on top of an “island“ made by inverting a smaller pan.,Carefully tuck the wick into the buffer wells.,At this point, the
10、gel has been denatured and neutralized and all the materials have been cut to size. The next step is placing the gel on the filter paper wick.,Once the gel has been denatured and neutralized, it is ready for Southern transfer.,Placing the gel on the Southern apparatus and preparing the nylon membran
11、e.,Once the gel has been denatured and neutralized, it is ready for Southern transfer. The gel is carefully inverted. When running a gel, the DNA is closer to the bottom. By inverting the gel, the DNA has less far to move through the gel when being transferred to the nylon membrane,The gel is invert
12、ed so the wells are now facing down, towards the filter paper wicks,The gel is centered on the filter paper wick, and any air bubbles are gently pushed out,Meanwhile, the nylon membrane is prepared by wetting it in distilled water.,The membrane is separated from the protective paper,Once the membran
13、e is wet, it is ready to place on the gel.,Assembling the Southern apparatus,The nylon membrane is the first item to be placed on the gel.,It is critical to line up the membrane and the gel. Once the membrane is on the gel, transfer starts almost immediately.,Lay down the membrane in a smooth motion
14、, either from the top down or from the center going out. Then smooth out any air bubbles that may be trapped between the membrane and the gel,The next step is applying the parafilm “mask“ around the perimeter of the membrane. Cover enough of the membrane so the parafilm stays in place, but not enoug
15、h to block any DNA lanes,Once the parafilm has sealed the nylon membrane, the two filter paper wicks are centered on top of the membrane. Finally, a stack of paper towels is put on top of the filter paper to encourage the capillary action,The finished Southern apparatus and the results,The completed
16、 Southern transfer apparatus. The DNA will transfer overnight. The membrane is removed, washed, and baked at 70C. The final procedure is developing the nylon membrane using nonradioactive probe detection.,The Southern gel, showing all the DNA bands from cleaving Lambda with different restriction enz
17、ymes. Since we know the sequence that is complimentary to our probe, we should be able to determine which fragment will be detected by the probe,The Southern blot shows only one band in each lane. This band corresponds to the band on the gel which contains the Lambda sequence that is complimentary to our probe. This sequence occurs only once in each digest and hence there is only one band per lane,
