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1、a newsletter for gene cloning, expression, and analysisvolume 10issue 5september 20036Analysis softwarefor Mac users10RNAi analysisnow faster and more efficient12Real-time PCR in a convenientsupermix format21Custom manufac-turing meets yourspecific needsSet your sights on a better reporter.Get incre
2、ased data accuracy with GeneBLAzerTechnology1 800 955 6288Expressions 10.5new product2unambiguous resultsWith GeneBLAzerTechnology, youll seesimple, unambiguous results. In the absenceof reporter activity, cells emit green fluores-cence; in the presence of reporter activity,they emit blue fluorescen
3、ce (figure 1). UsingGeneBLAzerTechnology in your reporterassays means youll get: Sensitive detection in live cells to increaseexperimental versatility and accuracy Cell sorting capabilities to isolate popula-tions of cells or to look for rare events Ratiometric readouts to normalize forexperimental
4、variables such as cell num-ber, providing increased data accuracyEasily monitor expression of genes andpathwaysand obtain the most accurateassay results.ratiometric readouts to reduce artifactsData from GeneBLAzerReporter Assays istypically expressed as a ratio between theamount of blue fluorescence
5、 and green fluo-rescence. The ratiometric nature of this dataincreases the reliability and robustness ofthese assayseven when miniaturized to384- or 1536-well plates. Thats becauseratiometric measurements normalize forexperimental differences in cell number, size,probe concentration, and instrument
6、variabil-ity. For example, you may produce the fol-lowing results from a transient assay experi-ment using a reporter to detect activation ofa kinase in response to several compounds: Using a non-ratiometric reporter:readoutWell 1Control5000Well 2Compound A10000Using the GeneBLAzerratiometricreporte
7、r technology:blue green readoutreadoutratioWell 1Control500010005Well 2Compound A1000020005In the example shown above, perhaps thecells in Well 1 did not plate well and aremore sparse than those in Well 2. Using anon-ratiometric reporter, it appears thatCompound A activates the kinase to agreater ex
8、tent than the Control does.GeneBLAzerTechnology normalizes forplating differences, so you can see that thereis no difference between Compound A andthe Control. Ratiometric data withGeneBLAzerprovides increased accuracyfor data pointsgiving you better results. BLAzing fast vectorsTwo types of vectors
9、 allow you to takeadvantage of GeneBLAzerTechnology inyour experiment: GeneBLAzerfusion vec-tors and a pGeneBLAzerpromoterless vec-Increased data accuracy in reporter assays with GeneBLAzerTechnologyive cell reporters are used to assess a variety of different cellular readouts, from pathwayelucidati
10、on to promoter bashing experiments. Choosing the best reporter for readouts isessential to obtaining accurate results. GeneBLAzerTechnology uses fluorescence resonanceenergy transfer (FRET) to provide the ideal reporter and get you the most accurate results. LGeneBLAzerTechnologycontinued on page 3f
11、igure 1 - FRET-based dual fluorescence provides increased data accuracyfigure 2 - FRET enables sensitive live cell detectionBlue cells indicate reporter activity; green cells indicate no activity for unambiguous detection. 409 nmFRET520 nmCCF2OOClClOOOOOOO OHNOSNOOHSOOOOOHNHNOONSOOOSOOOOO-lactamase4
12、47 nm409 nmUncleaved CCF2 emits green lightCleavage of CCF2 by -lactamase results in emission of blue lightAdapted with permission from Science, Vol. 276, p. 85 (January 2, 1998). 1998.-lactamase- Hydrolyzed ProductCCF2 SubstrateWavelength (nm)400450500550600650Relative Fluorescence IntensityEmissio
13、n peaks of cleaved and uncleaved CCF2Expressions 3tor (table 1). GeneBLAzerfusion vectorsenable you to easily fuse any gene to amammalian-optimized -lactamase gene(bla(M) so you can design your ideal assay.pGeneBLAzer, a promoterless vector, allowsyou to test the promoter activity of anyDNA sequence
14、. BLAze your research pathGeneBLAzerTechnology provides sensitivedetection in live cells and accurate, ratio-metric data making it the ideal technologyfor pathway analysis, cellular assays, andpromoter studies. Find out more today.These products may be covered by one or more Limited Use Label Licens
15、es (See the Invitrogen catalog or web site, ). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.GeneBLAzerTechnologyProductFeaturesChoose when:GeneBLAzerC-terminal Fusion of C-terminus of your gene to -lactamase You need high-level express
16、ion of a C-terminal fusion to -lactamaseGatewayFusion Kit for High-level expression from the CMV promoter You have an existing entry clone or UltimateORF CloneIn Vivo Detection attR sites for efficient recombination with a Gatewayentry cloneCat. no. 12578-043GeneBLAzerN-terminal Fusion of N-terminus of your gene to -lactamase You need high-level expression of a N-terminal fusion to -lactamaseGatewa
