摘要:MediaNeed LB plates (9 cm or 14 cm)to plate the library and do secondary screens.I typically screen about 500,000-1 million plaques for a primary cDNA screen.This fits on 11-22 14 cm plates.I do the s[阅读全文:]
摘要:Buffers and gel solutions Long Ranger: we started using this in early 1995. Great stuff; the best thing is that the gels are not sticky after drying, even without removal of the urea. Long Ranger Gel [阅读全文:]
摘要:载体（vector）是指运载外源DNA有效进入受体细胞内的工具。载体同外源DNA在体外重组成DNA重组分子，在进入受体后形成一个复制子，即形成在细胞内能独自进行自我复制的遗传因子。因此，作为载体应该满足以下几方面的要求：①有[阅读全文:]
摘要:1. For double-stranded DNA templates:a. Denature template: 10ml DNA (5-10g or alkaline lysis mini prepDNA)8ml ddH2O 2ml 2N NaOH -incubate 30' at 37℃.b. Dry-ice precipitate: +10ml 4M NH4OAc100ml EtOH[阅读全文:]
摘要:This method is after Marchuk,D.,et al.,1991,Nucl.Acids Res.19(5),pp1154. You will need:10 x Taq buffer (Promega)Taq Polymerase (Promega)Phenol/chloroform mix100mM dTTPTE bufferAbsolute ethanol70% etha[阅读全文:]
摘要:所谓亚克隆就是对已经获得的目的DNA片段进行重新克隆，其目的在于对目的DNA进行进一步分析，或者进行重组改造等。亚克隆的基本过程包括：(1)目的DNA片段和载体的制备；(2)目的DNA片段和载体的连接；(3)连接产物的转化；(4)[阅读全文:]
摘要:问题可能的原因建议转化后无克隆菌产生转化过程有问题或感受态细胞失活可通过转化pUC18/19等未切割的可用于抗性筛选的质粒，检测感受态细胞的转化效率和转化操作是否正确插入对照DNA片段的阳性率低 10×快速连接[阅读全文:]
摘要:1) The ligation mixture contains the following:vector (~100 ng) insert (equimolar or 2 or 3 X molar concentration of vector) water added to 18 µl2) Heat the mixture at 45 ℃ for 5 min. to melt [阅读全文:]