以番鸭脑垂体为材料,用Trizol法提取总RNA,用Superscipt Ⅱ-RT合成第一链cDNA,RNase H和DNA Polymerase合成第二链cDNA,最后用EcoR Ⅰ/Mse Ⅰ分步酶切cDNA各2h,经T4连接酶合成双链cDNA,预扩增反应后产物稀释40倍进行选择性扩增反应.经1％琼脂糖电泳及6％变性聚丙烯酰胺凝胶电泳检测的结果显示,番鸭脑垂体总RNA的提取及cDNA-AFLP预扩增和选择性扩增的效果良好.番鸭cDNA-AFLP反应体系的建立为番鸭就巢的分子机理研究奠定了基础.%The cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis system of muscovy duck was established u-sing pituitary as materials. The first single strand cDNA was gained through the reverse transcription from the total RNA. The second strand cDNA was synthesized by using Rnase H and DNA polymerase I DNA. Double-stranded cDNA was digested completely after 2 h digestion by EcoR Ⅰ and Mse Ⅰ step by step, and ligated to two adapters by T4 DNA ligase. The pre - amplification diluted to 40 times for selective amplification. The results of 1% agarose gel electrophoresis and 6% denaturing polyacrylamide gel electrophresis (PAGE) indicated that the RNA extraction, cDNA-APLP pre-amplification and select-amplification by above procedures were effective. The cDNA-AFLP analysis system was established successfully, which provided platform for molecular mechanism of broodiness of muscovy duck.
展开▼
