Description:
CLIP-Cell™505isagreenfluorescentsubstratethatcanbeusedtolabelCLIP-tag™fusionproteinsinsidelivingcells,oncellsurfacesorinvitro.Thiscell-permeablesubstrate(BC-505)isbasedontheDyomicsdyeDY-505andissuitableforstandardfluoresceinfiltersets.Ithasanexcitationmaximumat504nmandemissionmaximumat532nm.Thispackageincludes50nmolofCLIP-Cell505substrate,sufficienttomake10mlofa5µMCLIP-tagfusionproteinlabelingsolution.TheCLIP-tagproteinlabelingsystemenablesthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest.CLIP-tagisaproteintagbasedonhumanO6-alkylguanine-DNAalkyltransferase(hAGT).CLIP-tagsubstratesarederivativesofbenzylcytosine(BC).Inthelabelingreaction,thesubstitutedbenzylgroupofthesubstrateiscovalentlyattachedtothereactivecysteineofCLIP-tagformingastablethioetherbond. AlthoughCLIP-tagisbasedonthesameproteinasSNAP-tag®,thebenzylcytosinesubstratesformaseparateclassofsubstrates,differentfromthebenzylguaninesubstratesrecognizedbySNAP-tag.CLIP-tagandSNAP-tagcanbeusedfororthogonalandcomplementarylabelingoftwoproteinssimultaneouslyinthesamecells.
Therearetwostepstousingthissystem:subcloningandexpressionoftheproteinofinterestasaCLIP-tagfusionwiththeCLIP-tagfusionproteinsisdescribedinthedocumentationsuppliedwithCLIP-tagplasmids.Thelabelingofthefusionproteinsisdescribedinthisdocument.
CellswerelabeledwithCLIP-Cell505(green)for60minutesandcounterstainedwithHoechst33342(blue).


Notes:
Storage:CLIP-Cell505shouldbestoredat-20°C(longterm)orat4°Cinthedark(shortterm,lessthan4weeks).Protectthesubstratefromlightandmoisture.Withproperstorageat-20°Cthesubstrateshouldbestableforatleastthreeyearsdryor3monthsdissolvedinDMSO.ebiomall.com
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http://medgen.ugent.be/rtprimerdb/
在分子生物学中,探针是根据碱基互补的原理,用来与特定的DNA片段做杂交以对特定的DNA片段进行检测,如Southern杂交
引物是用来扩增DNA序列的,因为核苷酸必须要连接在3-OH上才能够合成延伸,引物就是用来提供3-OH的。
只要是符合要求的DNA片段都可以用来做探针和引物,引物是用来扩增DNA序列,探针用来与特定的DNA片段做分子杂交的
PCR即聚合酶链式反应是一种用于放大扩增特定的DNA片段的分子生物学技术,它可看作是生物体外的特殊DNA复制,PCR的最大特点,是能将微量的DNA大幅增加。
其原理为:
DNA的半保留复制是生物进化和传代的重要途径。双链DNA在多种酶的作用下可以变性解旋成单链,在DNA聚合酶的参与下,根据碱基互补配对原则复制成同样的两分子拷贝。在实验中发现,DNA在高温时也可以发生变性解链,当温度降低后又可以复性成为双链。因此,通过温度变化控制DNA的变性和复性,加入设计引物,DNA聚合酶、dNTP就可以完成特定基因的体外复制。
但是,DNA聚合酶在高温时会失活,因此,每次循环都得加入新的DNA聚合酶,不仅操作烦琐,而且价格昂贵,制约了PCR技术的应用和发展。
耐热DNA聚合酶--Taq酶的发现对于PCR的应用有里程碑的意义,该酶可以耐受90℃以上的高温而不失活,不需要每个循环加酶,使PCR技术变得非常简捷、同时也大大降低了成本,PCR技术得以大量应用,并逐步应用于临床。
PCR技术的基本原理类似于DNA的天然复制过程,其特异性依赖于与靶序列两端互补的寡核苷酸引物。PCR由变性--退火--延伸三个基本反应步骤构成:①模板DNA的变性:模板DNA经加热至93℃左右一定时间后,使模板DNA双链或经PCR扩增形成的双链DNA解离,使之成为单链,以便它与引物结合,为下轮反应作准备;②模板DNA与引物的退火(复性):模板DNA经加热变性成单链后,温度降至55℃左右,引物与模板DNA单链的互补序列配对结合;③引物的延伸:DNA模板--引物结合物在72℃、DNA聚合酶(如TaqDNA聚合酶)的作用下,以dNTP为反应原料,靶序列为模板,按碱基互补配对与半保留复制原理,合成一条新的与模板DNA链互补的半保留复制链,重复循环变性--退火--延伸三过程就可获得更多的“半保留复制链”,而且这种新链又可成为下次循环的模板。每完成一个循环需2~4分钟,2~3小时就能将待扩目的基因扩增放大几百万倍。
看引物能不能特异性扩增,你可以检查一下扩增后的熔解曲线,一般单一的峰是特异性引物,如果有双峰那就是扩出来了杂带
It is designed to address the challenge of pathogen detection, species identification and taxa discrimination using TaqMan and molecular beacon assays. With ClustalW multiple sequence alignment at its core, AlleleID can help design universal or taxa/species specific primers and probes. All major qPCR assays including molecular beacons, TaqMan, FRET or SYBR Green are supported. It uses BLAST search to assure specificity. To assure assay success, AlleleID uses template folding to avoid locating primers in the secondary structures of the template.
With AlleleID, you can avoid gDNA by designing primers/probes across exon/exon boundaries. The intron exon regions are identified by interpreting GenBank annotations or by specifying them manually.
To get started quickly with AlleleID, please download and listen to the multi-media tutorial:
http://www.PremierBiosoft.com/DATAFILES/AL100Tour.exe.
Then please download the demo version of the program from:
http://www.premierbiosoft.com/DATAFILES/AlleleIDTour.exe

