Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, more commonly referred to as DAPA, is a potent (Ki=10-7M) and specific synthetic thrombin inhibitor (1). Of special interest are the unique fluorescent properties contributed by the dansyl moiety of DAPA. When bound to thrombin, the fluorescence intensity and lifetime of the dansyl moiety are increased three fold. The enhancement of fluorescence intensity, coupled with its inhibitory properties, have made this compound extremely useful for studies involving thrombin generation (2-7). In addition to an increase in fluorescence intensity and lifetime, there is a decrease in depolarization of the excitation signal which also supports fluorescence polarization studies.
The inhibitor properties of DAPA alone have been useful in a variety of applications. DAPA is routinely used as a protease inhibitor during the isolation of proteins that are susceptible to thrombin cleavage. Furthermore, by eliminating the feedback of thrombin activity during prothrombin activation, the isolation of reaction intermediates such as fragment 1.2, and meizothrombin have been made possible (7).
DAPA is synthesized according the procedure described by Nesheim, et al. (1). HPLC, TLC and spectral analyses are used to determine the purity of the final product. DAPA is supplied in water. When properly stored at -20°C and protected from light, the compound is stable for many years.
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我做的双酶切反应，酶分别是宝生物的BamHI和XhoI，双酶切体系是：载体及目的片段分别是30ul，通用buffer4ul，XhoI、BamHI各2ul，无核酶水2ul，总体积40ul。载体和目的片段都是经37度酶切6h。载体上面这两个酶切位点的距离是6个碱基，目的片段是由pcr反应获得的，从puc19中p出来的，两端带有这两种酶的酶切位点，酶切完成后，做连接反应，体系如下：目的片段（全长1441bp）4ul，无核酶水3ul，10*T4DNA连接酶缓冲液1ul，载体1ul，T4DNA连接酶1ul，总体积10ul。16度过夜。之后摇菌送沉菌测序结果回来，送了5管一个都不对。重复实验两次还是不对！
之后改为先用BanHI单切载体，体系如下：载体17ul，酶1ul，buffer2ul，总体积20ul，30度切6小时后，Promega纯化试剂盒直接纯化，完了之后再用XhoI，37度酶切6小时，体系如上，再次用promega纯化试剂盒纯化，目的片段用双酶切体系酶切，之后做连接反应，之后铺板，挑取菌落共10个，摇菌13小时，取菌液做pcr鉴定，结果10个菌落全是引物2聚体，跑出来的条带都是100bp左右。
各位老师这是怎么回事呢？为什么连接不上呢？小弟先谢过各位大哥了！
配后是否应该分装,因为不能反复冻融?
多谢!
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【】可以这样输入，智能ABC，输入“v1”，后翻3页，就可以看见了，或用紫光拼音中文输入法，输入方括号就是粗体的中括号，实在不行请复制本公告中【】
求问酶切体系为10ul,内切酶1ul,37℃水浴45min，不知道DNA产物是否完全被切开了？
所用内切酶信息如下
感谢赐教~！
同一管dna有的酶可以切开，比如dra1，ecoR1,Hind111
有的酶又完全不能切动，如BamH1，sac1，xba1，xho1，pst1
请问高手这是什么原因？?
