M. tuberculosis Gyrase
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M. tuberculosis Gyrase (HIS)
Mycobacterium tuberculosis gyrase is a bacterial type II topoisomerase which can introduce negative supercoils into DNA. It is a target antibacterial drugs and can be used for screening potential novel antibacterial compounds. It is prepared by overexpressing the subunits with C-terminal polyhistidine tags in E. coli and then purified by affinity chromatography.
It is supplied as an A2B2 complex in Dilution Buffer. Store at -80 °C.
All enzyme is supplied with 5X concentrated Assay Buffer and Dilution Buffer which are also available separately. 1 U of gyrase will supercoil 0.5 µg relaxed pBR322 DNA in 30 minutes at 37 °C.
See technical documents below for more detailed information and lot specific activities.
Technical Documents
M. tuberculosis Gyrase (HIS) Supercoiling Assay Kits
Kits are available containing everything required to perform supercoiling reactions and to test inhibitors. They contain M. tuberculosis gyrase, relaxed DNA substrate and the Assay and Dilution buffers.
Technical Documents
M. tuberculosis Gyrase (HIS) Decatenation Assay Kits
These contain the M. tuberculosis gyrase and the kDNA substrate in addition to the Assay and Dilution buffers for decatenation reactions. 1 U of M. tuberculosis gyrase will decatenate 200 ng of kDNA when incubated in 1X Assay buffer in a total reaction volume of 30 µl at 37°C for 30 minutes.
Technical Documents
M. tuberculosis Gyrase Assay Kits for Cell Extracts
These kits are designed for assaying cell extracts containing M. tuberculosis gyrase and partially purified fractions and contain relaxed DNA substrate, Assay buffer, Dilution buffer and control supercoiled DNA.
Technical Documents
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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
配后是否应该分装,因为不能反复冻融?
多谢!
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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??
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!
感谢赐教~!