地高辛标记的探针和膜原位杂交法筛选cDNA文库

应用地高辛标记探针方法、筛选噬菌体ｃＤＮＡ文库并克隆ＣＮＴＦ基因，将文库以１０００个／皿的噬斑密度铺皿，待形成肉眼可见的噬斑后覆盖尼龙膜进行原位转移，再生变性、中和、交联等处理。ＰＣＲ法扩增出ＣＮＴＦ探针，随机引物法进行地高辛标记，与处理后的尼龙膜杂交，挑出阳性斑进行第二轮低噬斑密度筛选，得到ＣＮＴＦ单克隆基因片断，此法具有实验操作简单、周期短、探讨针保存时间长，并可反复使用的特点。

THE MEANS OF SCREENING cDNA LIBRARY BY DIG-LABLED PROBE AND PLAQUE HYBRIDIZATION

This article introduces the means of screening cDNA library and coloning CNTF gene by DIG-labled probe, culturing phage for the density of 10000 pfu/ware, transferred plaque to a nylon membrane when the plaque could be seen by eyes. Then denatured, neutralized, crosslinked the transferred DNA in the Membrane. After amplifying CNTF probe by PCR, labling the probe with DIG by random primers, We used the probe to hybridize with the treated membrane, then chosed the positive plaque for the second screening and obtained the monocolony plaque of CNTF gene fragment.