Lufkin Thomas
CSH Protoc. 2007 Nov 1;2007:pdb.prot4822. doi: 10.1101/pdb.prot4822.
INTRODUCTIONThis is the first of two protocols describing how to perform in situ hybridization on whole mouse embryos. Here we describe how to fix and permeabilize embryos with detergent and how to synthesize single-stranded RNA probes (riboprobes) in vitro using UTP labeled with the plant steroid digoxigenin (DIG-11-UTP). Antisense digoxigenin-labeled riboprobes are synthesized as run-off transcripts from linearized plasmid templates using bacteriophage RNA polymerases (T3, T7, SP6) under standard conditions. The probe is precipitated with ethanol in the presence of NaCl, and after resuspension in DEPC-treated H(2)O is stable at -20°C for many months. Probes ranging from 250 to 1500 bp have been used successfully. The optimal probe concentration is in the 100 ng to 1 μg/mL range and should be determined empirically.
引言
这是描述如何对整个小鼠胚胎进行原位杂交的两个方案中的第一个。在这里，我们描述了如何用去污剂固定和通透胚胎，以及如何使用用植物类固醇地高辛配基（DIG-11-UTP）标记的UTP在体外合成单链RNA探针（核糖探针）。在标准条件下，使用噬菌体RNA聚合酶（T3、T7、SP6）从线性化质粒模板合成反义地高辛配基标记的核糖探针作为径流转录本。探针在NaCl存在下用乙醇沉淀，重悬于经DEPC处理的H₂O中后，在-20°C下可稳定保存数月。已成功使用250至1500 bp的探针。最佳探针浓度在100 ng至1 μg/mL范围内，应根据经验确定。
