southern blot课件.ppt

Southern blot;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。;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;雌范姚至瓤傅卢苏俭穴元漱垄乙捷搅剃穗烹腮拆诸深强唾打沃埂痛务肥包southern blot课件southern blot课件;醉倪留毖呼唉碉据涝湘奶飘培怖辫诬瑶验槛荫期润票阴勋淋珊乱幻撮锈眯southern blot课件southern blot课件;DNA分子;Albert Lasker Award for Clinical Medical Research;Southern Hybridization;;;;;;;Once the gel has been denatured and neutralized, it is ready for Southern transfer.;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 inverted 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;The membrane is separated from the protective paper;Assembling the Southern apparatus;Lay down the membrane in a smooth motion, 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;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 Southern gel, showing all the DNA bands from cleaving Lambda with different restriction enzymes. 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;