Mammalian homeo box genes.
Mammalian homeo box genes.
复制标题
哺乳动物同源盒基因。
DOI：
10.1101/sqb.1985.050.01.035
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Fienberg,A
中科院分区：
文献类型：
--
作者：
Ruddle,FH;Hart,CP;Awgulewitsch,A;Fainsod,A;Utset,M;Dalton,D;Kerk,N;Rabin,M;Ferguson-Smith,A;Fienberg,A
材料和方法基因组文库筛选。基因组文库（克隆到 Charon 28 中的 BALB/c 小鼠胚胎 DNA）由 P. Leder（哈佛医学院）提供，并按先前所述进行筛选（McGinnis 等人，1984a）。使用果蝇触角足和 Ultrabithorax 同源框探针一式两份筛选噬菌斑 (104)（有关说明，请参阅 McGinnis 等人 1984b）。Southern 印迹杂交。用限制性核酸内切酶消化基因组DNA(15μg)或噬菌体DNA(0.25μg)并在0.8507μg琼脂糖Tris-硼酸盐凝胶中进行电泳。根据 Southern (1975) 的程序将 DNA 转移到硝化纤维素上。杂交在 6• SSC、2 x Denhardt's、0.1070 SDS 和 150 gg/ml 变性超声处理鲑鱼精子 DNA 中进行，如果是基因组印迹，则使用 10070 硫酸葡聚糖。在 [32p] dCTP 存在下将杂交探针进行切口平移，使其比活性约为 108 cpm/# g，并以 10 ng/ml 的杂交混合物添加。孵育12-16小时后，首先将滤膜在2℃和0.1070 SDS中在65℃下洗涤30分钟，然后在65℃下再次在0.1℃和0.1070 SDS中洗涤30分钟。
MATERIALS AND METHODSGenomic library screening. The genomic library (BALB/c mouse embryo DNA cloned into Charon 28) was provided by P. Leder (Harvard Medical School) and screened as described previously (McGinnis et al. 1984a). Plaques (104) were screened in duplicate with the Drosophila Antennapedia and Ultrabithorax homeo box probes (for a description, see McGinnis et al. 1984b).Southern blot hybridization. Genomic DNA (15 gg) or phage DNA (0.25/~ g) was digested with restriction endonuclease and electrophoresed in 0.8507 o agarose Tris-borate gels. DNA was transferred to nitrocellulose according to the procedure of Southern (1975). Hybridizations were done at 65~ in 6• SSC, 2 x Denhardt's, 0.1070 SDS, and 150 gg/ml of denatured sonicated salmon sperm DNA, and in the case of genomic blots, 10070 dextran sulfate. The hybridization probes were nick-translated in the presence of [32p] dCTP to a specific activity of approximately 108 cpm/# g and added at 10 ng/ml of hybridization mix. Following a 12-16-hour incubation, the filters were first washed at 65~ in 2• and 0.1070 SDS for 30 minutes and then washed in 0.1• and 0.1070 SDS again at 65~ for 30 minutes.