园艺的实.验手册.docVIP

实 验 手 册 华中农业大学作物遗传改良国家重点实验室 细胞工程分室柑桔课题组 二OO二年七月●武汉 目 录 前 言 1 组 织 培 养 2 1、培养基母液的配制 3 2、柑桔胚性愈伤组织的诱导 5 3、柑桔原生质体操作 6 4、柑桔胚性愈伤组织和茎尖的超低温保存 11 5、微嫁接 13 6、花粉的制备和保存 13 分子生物学 14 1、DNA提取和检测 15 2、Southern blotting 19 3、Northern blotting 27 4、AFLP 标记 30 5、SSR操作步骤 36 6、ISSR操作步骤 37 7、根癌农杆菌介导柑橘愈伤组织遗传转化 38 8、PCR 产物的TA克隆 41 9、植物RNA的分离和纯化（异硫氰酸胍法） 47 10、GISH分析 49 仪 器 使 用 51 1、色彩色差计CR-300的使用流程及注意事项 52 2、细胞流式仪（PA）操作规程 55 前 言 We come here for the common interest. Every year we put our heart into the research so as to find something. But for a beginner it is of great difficulty for him to initiate the first step. Much time should be spent on the protocol reading, document retrieval and method optimization. In order to, to some degree if not completely, avoid this tedious, time-consuming and baffling period the students in the Citrus Research Group of Cell Engineering Division of National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, come up with the idea of summarizing their research protocols and ideas, which is what you will see now. I think this will be of great help for those who want to do the similar work shown herein. Though you can see the protocols in other references you cannot get every detail for the experiment because the introduction there is very sketchy. On the contrary you can see the details from the students’ personal generalization in this manual. The present manual covers many horizons of biological sciences, ranging from tissue culture to molecular markers that are state-of-the-art topics for the time being. In addition some introduction of machine operations are included so that those who want to use the instrument can benefit from this manual. Thanks should be extended to Prof. Xiuxin Deng for his kind offer of the financial support for the smooth publication of this manual. In the meantime gratitude should be given to all of the postgraduate students who have been working, are working here. Without their hard work we would not