S. aureus Topoisomerase IV
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S. aureus Topoisomerase IV
Topo IV (from Staphylococcus aureus) is prepared by overexpressing the subunits in E. coliand purifying them by methods developed in-house.
The subunits are purified to >95% purity as judged by SDS-PAGE. The topo IV is supplied as a heterotetramer complex in Dilution buffer.
It is recommended that the enzyme is aliquoted to avoid repeated freeze-thaw cycles. Store at -80ºC.
All enzyme is supplied with 5X concentrated Assay Buffer and Dilution buffers which are also available separately.
See technical documents below for more detailed information and lot specific activities.
Technical Documents
S. aureus Topoisomerase IV Relaxation Assay Kits
These contain S. aureus topo IV and the supercoiled DNA substrate in addition to the Assay and Dilution buffers for relaxation reactions. 1 U of topo IV will relax 0.5 µg supercoiled pBR322 DNA in 30 minutes at 37°C.
Technical Documents
S. aureus Topoisomerase IV Decatenation Assay Kits
These contain S. aureus topo IV and the catenated kDNA substrate in addition to the Assay and Dilution buffers for decatenation reactions. 1 U of topo IV 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
S. aureus Topoisomerase IV Cleavage Assay Kits
These kits are designed specifically for cleavage reactions. They contain S. aureus topo IV enzyme, supercoiled pBR322 DNA substrate and the Assay and Dilution buffers required for DNA cleavage reactions in addition to linearised pBR322 marker.
Cleavage specific enzyme available separately on request.
Technical Documents
S. aureus Topoisomerase IV Assay Kit for Cell Extracts
These kits are designed for assaying cell extracts and partially purified fractions containing over-expressed S. aureus topo IV and contain supercoiled DNA substrate, Assay buffer, Dilution buffer, control relaxed DNA and stop buffer/loading dye.
Technical Documents
S. aureus Topoisomerase IV ATPase kit
These kits can be used to test the effects of potential ATPase inhibitors. For example, the coumarin drugs such as novobiocin inhibit the action of topoisomerase IV by competitively inhibiting the hydrolysis of ATP thus preventing supercoiling.
These assays are microtitre plate-based and thus large numbers of compounds can be screened in a relatively short period of time. They also continuous assays which can provide more information than an end point assay.
Technical Documents
High / Medium-Throughput Assay Kit - S. aureus Topoisomerase IV
The kit is supplied with sufficient S. aureus topo IV enzyme, plasmid DNA substrate, buffers and other assay components* for 100 assays. The enzyme is supplied at a concentration of 10 U/μl in Dilution Buffer. The kit is also supplied with sufficient wash buffers for one 96-well plate. These buffers are supplied as 20X concentrates and must be diluted with ultra pure water prior to use.
More information about this assay can be found on the "Services" page under "High/Medium Throughput Assay".
Kit issued with limited licence for individual use only.
Patent held by Inspiralis Ltd., Norwich, Norfolk, UK. (Patent No. GB0424953.8, US7838230)
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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【】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【】
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~!