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1. T ae Illustration inspired by the art of Pablo Picasso 1882 1973 PCR in a plate It s that easy e 96 well PCR in a fraction of the time why spend all of your time aliquotting e Higher sensitivity fidelity and yields for all of your high through put PCR applications e 96 well plates that are compatible with all major PCR block and robotics manufacturers Tired of liquid handling bottlenecks Experiencing higher than acceptable fail rates Fed up with optimizing a low performance Taq polymerase for high performance applications Relax high throughput PCR is now easier and faster Please see the BD Sprint Kits N otice to Purchaser on page 1 BD Biosciences Clontech ww w bdbiosciences com United States Canada Europe Japan Introducing the revolutionary new BD Sprint Advantage 96 Plate K 1950 1 with everything you need to complete 96 PCR reactions except for water your primers and your DNA template Each well of the BD Sprint Advantage 96 Plate contains a complete lyophilized master mix comprised of BD TITANIUM Tag BD TaqStart Antibody a proofreading enzyme for increased fidelity dNTPs and an optimized PCR buffer To use the plate simply resuspend the master mix in your diluted primers and template DNA 25 ul total then go directly to PCR High performance high throughput PCR in a plate it s just that easy Asia Pacific Latin America Caribbean 82. 662 643 1381 Delta Laboratory Co Ltd Tel 66 2 530 7341 4 Fax 66 2 559 3365 TURKEY BD Italia SPA Tel 39 02 4824 0242 Fax 39 02 4820 3336 UNITED KINGDOM BD Biosciences UK Tel 1865 781688 Fax 1865 781627 VENEZUELA BD de Venezuela Tel 58 212 443 6411 x 248 Fax 58 212 442 4477 TP25272 08 01 02 BD Biosciences Clontech 1020 East Meadow Circle Palo Alto CA 94303 4230 Toll Free 877 232 8995 Tel 650 424 8222 Fax 650 424 0133 BD BD Logo and all other trademarks are the property of Becton Dickinson and Company 2002 BD Environmentally produced 1906 33C
3. any vector No insert 1 kb PCR amplify target gene with primers containing 15 bp overhangs homologous to ends of linear vector Linearized pDNR Dual Add BD In Fusion Enzyme gt 3 Strand displacement by BD In Fusion Enzyme pDNR Dual 30 min BD In Fusion Enzyme captures DNA ends at 25 C and fuses PCR product to the vector pDNR Dual A BD In Fusion Enzyme Wa 5 Primer Sequence Transform E coli 3 Primer Sequence Figure 1 The BD In Fusion cloning method 2 kb 3 kb 4 kb Figure 2 BD In Fusion Method efficiently clones a range of insert sizes BD In Fusion Cloning was performed using 100 ng linear vector and 50 ng of each of the PCR products indicated 1 ul of each 20 ul reaction was then transformed into BD Fusion Blue competent cells After 1 hr of outgrowth 1 10 of the volume of each transformation was plated on BD CLONdisc plates BD Biosciences Clontech www bdbiosciences com Clontechniques October 2002 New Products BD In Fusion PCR Cloning KIt continued Table I BD In Fusion is the most flexible and comprehensive cloning system available BD In Fusion PCR Cloning Kit Directional J Primer extension required A Reaction time 10 30 min Blue w hite screening A Proofreading polymerase A Ligase not required J Gene transfer capabilities BD Creator Systems High efficiency cloning of J long fragments In addition virtually any PCR fragmen
4. you with consistent gene coverage and great flexibility use it for data normal ization with any array and any labeling method Easily compare microarray results Our Reference Total RNA allows you to compare data sets from different micro array experiments Simply hybridize a probe made with our Reference Total RNA to a microarray each time you per form an experiment and then normalize your data to the Reference Total RNA Because we furnish you with enough Reference Total RNA for up to 80 microarray experiments you can compare results over a series of experiments Our Reference Total RNA is the best approach to building gene expression databases in which you compare expres sion profiles from different tissue or cell line models To provide you with the best overall gene representation with the least variation in gene expression we made our Reference Total RNA using a combination of differ ent tissue sources Figure 1 RNA extracted from a range of different whole tissue sources is purified using our BD Premium RNA method Then the RNA from each tissue is pooled creating one master stock of high quality ultra pure Reference RNA that has a more even gene distribution than any individual tissue tested We have found that RNA from whole tissues shows higher overall expression with less variation than RNA from cell lines 1 The result is an RNA reference standard that consistently provides homogenous signa
5. 100 rxns K1916 2 BD Fusion Blue Competent Cells 22 tubes 5004 1 BD In Fusion Kit Components BD In Fusion Enzyme concentrate BD In Fusion Enzyme Dilution Buffer 10X BD In Fusion Reaction Buffer 10X BSA pDNR Dual linearized 1 1 kb Control Insert Related Products BD Advantage PCR Kits many BD Sprint Advantage 96 Plate K 1950 1 pLP CM V Acceptor Vector 8901 1 pLP EY FP C1 Acceptor Vector 6341 1 pLP EGFP C1 Acceptor Vector 6342 1 pLP ECFP C1 Acceptor Vector 6343 1 pLP LNCX Acceptor Vector 6344 1 pLP IRES2 EGFP Acceptor Vector 6345 1 pLP IRESneo Acceptor Vector 6346 1 pLP RevT RE Acceptor Vector 6347 1 pLP TRE2 Acceptor Vector 6348 1 pLP GADT7 AD Acceptor Vector 6349 1 pLP GBKT7 DNA BD Acceptor Vector 6350 1 pLP CM V M yc Acceptor Vector 6351 1 pLP PROTet 6xHN Acceptor Vector 6352 1 BD Creator Acceptor Vector Construction Kit K 1690 1 pLPS 3 EGFP Acceptor Vector 6360 1 pLP CM Vneo Acceptor Vector 6361 1 pLP CM V HA Acceptor Vector 6362 1 pLP BacPAK 9 Acceptor Vector 6211 1 pLP BacPAK 9 6xHN Acceptor Vector 6212 1 Clontechniques October 2002 11 New Products BD Creator BacPAK9 Shuttle Vectors Easy preparation of baculoviral shuttle constructs via Cre loxP recombination e BD Creator cloning is fast and efficient e Vectors for native expression of proteins under optimal folding conditions Tagged expressi
6. Acid Purification New NucleoSpin Blood and Virus Nucleic Acid Purification Kits 13 Cell Biology Destabilized DsRed Express and HcRed VectorS 00 eevee 14 BD Mercury TransFactor Profiling Kit Oncogenesis3 16 Technical Note BD Premium Total RNA Contains Virtually No Genomic DNA an Important Factor in RNA Quality 0 0 cece ees 8 Announcements New Products Coming SOON 1 ee eee 17 BD Biosciences Clontech products are intended to be used for research purposes only They are not to be used for drug or diagnostic purposes nor are they intended for human use Products may not be resold modified for resale or used to manufacture commercial products without written approval of BD Biosciences Clontech Clontechniques is published quarterly in January April July and October by BD Biosciences Clontech 1020 East M eadow Circle Palo Alto CA 94303 4230 USA Notice to Purchaser for BD Sprint Kits A license under U S Patents 4 683 202 4 683 195 and 4 965 188 or their foreign counterparts owned by H offmann LaR oche and F Hoffmann La Roche Ltd Roche has an up front fee component and a running royalty component The purchase price of this product includes limited non transferable rights under the running royalty component to use only this amount of the product to practice the Polymerase Chain Reaction PCR and related products described in said patents solely for the r
7. Premium Total RNA Determining genomic DNA effect Our next step was to determine the con tribution of genomic DNA contamina Clontechniques October 2002 Technical Note BD Premium Total RNA Contains Virtually No Genomic DNA an Important Factor in RNA Quality continued A BD Premium Total RNA Vendor A Total RNA Vendor S Total RNA kb M c os slc M spllc M Sp B BD Premium Total RNA Vendor A Total RNA Vendor S Total RNA kb M C SM Sp C SM Sp C SM Sp Figure 3 Positive RT PCR results from total RNA samples containing genomic DNA Panel A Firs strand cDNA was synthesized using BD PowerScript Reverse Transcriptase 8460 1 and 1 ug of total RNA PCR was subsequently performed using an aliquot of the RT reaction with primers for a CDNA fragment of the phospholipase A2 gene After 28 cycles RT PCR products were analyzed using agarose EtBr gel electrophoresis C colon SM skeletal muscle Sp spleen M 500 bp Molecular Ruler Bio Rad Laboratories 170 8203 Arrow denotes the 700 bp product Panel B Total RNA samples were used directly as a template for PCR omitting prior RT step using the same primer set After 35 cycles PCR products were analyzed using agarose EtBr gel electrophoresis Sample lanes are assigned asin Panel A tion to a standard RT PCR experiment We first performed a parallel RT PCR analysis of all total RNA samples using primers that amplify a cDNA fragment of the hou
8. Product Size Cat Mouse Universal Reference Total RNA 2x 200 ug 64118 1 Human Universal Reference Total RNA 2 x200 ug 64115 1 Reference 1 Control RNA for M icroarray Experiments April 2002 Clontechniques X VII 2 6 Clontechniques October 2002 5 6 New Products Cancer Profiling Array Il and Tissue Specific Cancer Profiling Arrays Obtain reliable gene expression data from a variety of cancer samples e Access many hard to obtain human tissues at an affordable price e Identify tumor specific markers tumor suppressor genes or potential drug targets e Generate statistically significant data for determining gene relevance In cancer BD Biosciences Clontech introduces a new addition to our line of Cancer Profiling Products The Cancer Profiling Array II contains 154 pairs of cDNAs generated from matched normal and tumor tissue samples from individual patients spotted side by side on a nylon membrane This new array includes 6 additional tissues not available on our Original Cancer Profiling Array so now you can seek tumor specific markers in a total of 19 tumor types at once M ost of these tumor types are represented by 10 patients allowing you to generate statistically significant data for your target gene in a single experiment Like our other Cancer Profiling Arrays this array is made using BD SMART technology and sample normalization so you ll obtain reliable accurate data when ide
9. TransFactor Kits are available in individ ual and profiling formats Individual Kits let you investigate a single transcription factor in depth Profiling Kits on the other hand enable you to screen the DNA binding activities of multiple fac tors involved in specific biological processes such as inflammation and oncogenesis Profiling plates Shown above are divided into six sets of color coded wells each set contains the cis acting DNA element for a specific transcription factor All TransFactor Plates consist of unique snap off wells so you can reconfigure the plates to fit your experimental design You may perform all 96 reactions at once or remove wells individual ly or in strips for use at a later time 1 2 1 Cos 7 CoCl 0 8 E Cos7 0 6 0 4 OD 655 nm 0 2 0 S S A Xe x cs L Figure 2 Transcription Factor Profiling with Oncogenesis 3 Cos 7 cells were treated with 0 15 mM CoCl for 23 hr Nuclear extracts were then prepared using the BD TransFactor Extraction Kit lt 2064 1 and assayed according to the protocol in the BD Mercury TransFactor Kits User Manual PT3594 1 Q N S TransFactor assays typically take 3 4 hours and are 10 times more sensitive than EM SA 1 2 The flexible 96 well format gives you the ability to compare multiple samples simultaneously Figure 2 You can even perform competition assays to assess binding specificity and to deter mine the key bases in the protein b
10. Using the BD Atlas Plastic Human 12K Microarray identifies gene expression in human colon total RNA The array was hybridized using a 33P labeled probe generated from human colon BD Premium Total RNA according to instructions outlined in the User Manual PT3591 1 that combines the high hybridization effi ciency of a cDNA fragment with a short oligonucleotide s ability to distinguish between homologous genes We use antisense hybridization to thoroughly test each oligonucleotide confirming its identity and ability to produce a strong hybridization signal O ligos that display weak hybridization signals or exhibit cross hybridization to other fragments are redesigned Without these tests greater than 25 of all oligos would be incapable of producing a unique and usable hybridization signal BD Biosciences Clontech is the only company performing this type of rigorous antisense testing giving you a microarray that delivers credible results Figure 2 Take advantage of a unique format BD Atlas Plastic M icroarrays offer an unparalleled combination of ease and efficiency Like nylon arrays BD Atlas Plastic M icroarrays require no special equipment for imaging just a standard phosphorimager And like glass arrays these plastic arrays are nonporous which greatly decreases nonspecific background and minimizes washing time The unique quality of the plastic material allows the printing of far more spots than on a nylon memb
11. a world wide non exclusive royalty free limited license to use this product for non commercial life science research use only Such license specifically excludes the right to sell or otherwise transfer this product or its components to third parties Any other use of this product will require a license from BD Biosciences Clontech Please contact our licensing hotline by phone at either 800 662 2566 or 650 424 8222 extension 7816 or by e mail at licensing clontech com For Profit entities that wish to use this product in non commercial or commercial applications are required to obtain a license from BD Biosciences Clontech For license information please contact our licensing hotline by phone at either 800 662 2566 or 650 424 8222 extension 7816 or by e mail at licensing clontech com This product is the subject of pending U S and foreign patents Clontechniques October 2002 Q 15 New Products BD Mercury TransFactor Profiling Kit Oncogenesis 3 A high throughput assay for detecting DNA protein interactions Analyze the DNA binding activity of multiple transcription factors simultaneously with one assay e Faster and more sensitive than gel shift assays e Flexible 96 well format A key step in the regulation of gene expression is the binding of a transcription factor to its cis acting DNA response element In the past researchers routinely measured such activities using an electrophoretic mobi
12. on gene expression With our Cancer Cell Line Profiling Array you will soon be able to quickly determine the effects of a wide variety of cancer treatments on your genes of interest This nylon array includes cDNA samples prepared from 26 different cancer cell lines that were each treated with 26 agents including chemotherapies stress inducers and radiation Simply hybridize a radiolabeled probe to assess a gene s expression in response to treatment to investigate its role in disease or to predict novel gene function based on the expression profile of known genes Eleven different tissue types are represented on the array to provide a broad sampling of different cancer types the time Ultra high throughput PCR in a fraction of Last Spring we launched our revolutionary BD Sprint Advantage 96 Plate for high throughput PCR Soon we will be going to the next level with our BD Sprint TITANIUM Taq 384 Plate This 384 well plate provides everything you need for PCR in lyophilized form and is ideal for genotyping and SNP studies as well as any other ultra high throughput PCR application in which sensitivity and yield are critical The TITANIUM Taq 384 Plate is compatible with all ultra high throughput PCR machines and robotic systems Simply resuspend the lyophilized mix with 10 ul of water containing primers and template and go directly to PCR and results Please see the BD Sprint Kits N otice to Purchaser on page 1 BD B
13. pHcRed1 DR The constructs were then transiently transfected into HeLa cells After overnight incuba tion cells were analyzed by flow cytometry using a BD FACSVantage SE at three separate times first to establish the baseline fluorescence second to measure the fold induction after 4 hours of treatment with 100 ng ml TNF a and third to measure down regulation 12 hours after withdrawing TNF a from the culture In this example HcRed1 DR was excited with a 568 nm laser line DsRed Express with a 488 nm line Panels B amp C Photomicrographs of cells transiently transfected with pNF B DsRed Express DR before Panel B and after Panel C induction The image was recorded with a Zeiss Axioskop using Chroma Technology Corp filters hg545 50X 580dcxr and hq630 60M Red fluorescence and rapid turnover heighten the sensitivity of your assays Destabilized fluorescent proteins have clear advantages over their long lived counterparts First when placed under the control of an inducible promoter destabilized variants exhibit a higher fold induction upon activation Figure 1 That s because the small amount of protein expressed in the uninduced state is rapidly degraded so the baseline fluorescence in the uninduced state is low and the lower the baseline the greater the sensi tivity of your assay Second with their long wavelength excitation maxima 14 BD Biosciences Clontech www bdbiosciences com destabilized red fluorescent prote
14. your BD Biosciences Clontech sales representative or visit www clontech com premium rna Supplies are limited so please inquire about availability Clontechniques October 2002 9 New Products 10 BD In Fusion PCR Cloning Kit Precise directional cloning of PCR products without restriction enzymes No restriction enzyme or ligase required e Compatible with the BD Creator System for immediate expression analysis e No A overhang requirement Use any thermostable polymerase for amplification e Robust performance easily clone up to 8 kb The BD In Fusion PCR Cloning Kit is designed for fast high throughput cloning of PCR products without the need for restriction enzymes ligase or blunt end polishing This kit includes our proprietary BD In Fusion Enzyme and pDNR Dual Donor Vector for generating precise directional constructs that are immediately ready for expression analysis with our BD Creator Gene Cloning amp Expression System The BD In Fusion PCR cloning method The BD In Fusion method consists of a simple 30 min benchtop incubation of the PCR product with the linearized oDNR Dual Vector followed by trans formation of E coli Figure 1 Optional blue white selection on X Gal plates can be used to screen out rare non lin earized vector background Although linearized pDN R Dual is provided the BD In Fusion enzyme action is universal and allows cloning of PCR products into
15. 1608 Fax 86 10 6418 1610 Genetimes Inc Tel 86 21 3424 0452 Fax 86 21 6469 3766 Gene Co Ltd Tel 8621 6495 1899 Fax 8621 6428 7165 COLOMBIA BD Colombia Tel 57 1572 4060 Fax 57 1572 4250 573 2908 CZECH REPUBLIC T A Interact s r o Tel 420 2 2481 0196 Fax 420 2 2231 4055 DENMARK BD Biosciences Denmark Tel 45 43 43 45 66 Fax 45 965 676 EASTERN EUROPE MIDDLE EAST AFRICA BD Biosciences EMA Tel 49 6221 305 161 Fax 49 6221 305 418 EGYPT m p t Medicopharmatrade Tel 202 749 3734 Fax 202 749 8311 FINLAND BD Biosciences Finland Tel 358 98 870 780 Fax 358 98 870 7817 FRANCE BD Biosciences France Tel 33 4 7668 3636 Fax 33 4 7668 3504 Ozyme Tel 33 1 34 602424 Fax 33 1 34 609212 GERMANY BD Biosciences Germany Tel 49 6221 3417 0 Fax 49 6221 303 511 GREECE Bio Sure Tel 301 973 5245 Fax 301 970 3736 HONG KONG BD ASIA Ltd Tel 852 2575 8668 Fax 852 2803 5320 HONG KONG Bio Gene Technology Ltd Tel 852 2646 6101 Fax 852 2686 8806 HUNGARY Novo Lab Bt Tel 36 1 281 3692 Fax 36 1 281 3763 INDIA BD India Private Ltd India Tel 91 124 638 3219 Fax 91 124 638 3225 ISRAEL Biological Industries Co Tel 972 4 9960595 Fax 972 4 9968896 ITALY BD Italia SPA Italy Tel 39 02 4824 0242 Fax 39 02 4820 3336 JAPAN BD Biosciences Japan Tel 81 3 5324 9609 Fax 81 3 5324 9636 KOREA BD Biosciences Korea Tel 82 2 3404 3773 Fax 8
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17. 77 232 8995 888 259 0187 32 53 720 211 81 24 593 5405 65 6861 0633 55 11 5185 9995 For country specific contact information vist www bdbiosciences com how_to_order For Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale BD BD Logo and all other trademarks are the property of Becton Dickinson and Company 2002 BD AD29849 BD Sprint Advantage A a re 96 Plate BD Biosciences Clontech Discovery Labware Immunocytometry Systems Pharmingen Clontechniques October 2002 Volume XVII No 4 Mk A NY About the Cover The cover shows an illustration designed for our Disease Profiling Arrays Illustration inspired by the art of Roy Lichtenstein 1923 1997 Clontechniques Editor Eric Machleder Production Coordinator Erin McLey Graphic Designer Janice McClure Contributing Editors Suzanne Canada Ph D Patricia Wong Ph D Louis Wollenberger Ph D IN THIS ISSUE Gene Expression Profiling BD Atlas Plastic Human 12K Microarray 0000s 2 BD Atlas Custom Array Printing Services 0000 cece aa 4 M ouse Universal Reference Total RNA cee eee 5 Cancer Profiling Array Il and Tissue Specific Cancer Profiling Arrays 6 Gene Cloning and Expression BD In Fusion PCR Cloning Kit 0 0 00 cee ees 10 BD Creator BacPAK9 Shuttle Vectors aaa ees 12 Tet System Approved FBS cc tte 17 Nucleic
18. CS2003 inquire BD Atlas Custom Nylon Array each TP1002 inquire Notice to Purchaser for BD Atlas Products The BD Atlas Array products sold by BD Biosciences Clontech are for research purposes only Certain isolated DNA sequences included on the BD Atlas Arrays may be covered by U S Patents Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using BD Atlas Arrays These products and the sequences of the polynucleotides thereon are intended to be used for the purchaser s own internal research pur poses only and may not be used for drug development or diagnostic purposes or for human use Using BD Atlas Glass Microarrays for dual color analysis on a single array in which at least two different samples are labeled with at least two different labels may require a license under one of the following patents U S Patent Nos 5 770 358 or 5 800 992 Affymetrix and U S Patent No 5 830 645 Regents of The University of California Getting organized Use our bioinformatics web site to manage your gene lists Along with the Virtual Array Builder our Bioinformatics home page provides a complete set of tools to help you compare your list of genes to our list of cDNA fragments and long oligos In the process you can separate your list into unique and non unique entries find corresponding LocusLink and Unigene ID numbers eliminate redu
19. D ROM PT 3593 CD User M anual PT 3591 1 Related Products e BD Atlas BD Atlas BD Atlas BD Atlas Plastic Human 8K M icroarray 7905 1 Plastic M ouse 5K M icroarray 7906 1 Plastic Rat 4K M icroarray 7909 1 Plastic M icroarray Trial Kit K 1845 1 BD Atlasimage 2 7 Software V 1214 1 BD AtlasN avigator 2 0 Software V 1221 1 BD Atlas Plastic Array H ybridization Box 7930 1 BD Atlas Plastic Printing Kit 1846 1 BD Atlas Custom Plastic Arrays CS2050 1 BD Atlas Custom Plastic H ybridization and Analysis CS2013 1 BD Atlaslmage Custom Analysis Service C S2002 Notice to Purchaser t Patent Pending The BD Atlas Array products sold by BD Biosciences Clontech are for research purposes only These products and the sequences of the polynucleotides thereon are intended to be used for the purchaser s own internal research purposes only and may not be used for diagnostic purposes or for human use Clontechniques October 2002 Q 3 New Products BD Atlas Custom Array Printing Services Design your own BD Atlas Array on line and let our experts do the rest e Print any gene on glass plastic or nylon e Choose from our extensive collection of human mouse and rat genes or add your own e Easy to use on line Virtual Array Builder and Gene Search tools H aving trouble finding a gene array to fit your needs M aybe you just want to foc
20. ar weight marker Lane 2 pLP BacPAK9 6xHN Acceptor Vector digested with Aat II Lane 3 Donor Vector digested with Aat II Lanes 4 11 recombinants from Cre reaction digested with Aat II Seven out of eight recombinants contain the correct insert Figure 2 Expression of Enhanced Green Fluorescent Protein EGFP and 6xHN tagged EGFP from BacPAK9 Shuttle Vector constructs in Spodoptera frugiperda Sf21 cells pLP BacPAK9 and pLP BacPAK9 6xHN were used to generate pLP BacPAK9 EGFP and pLP BacPAK9 6xHN EGFP respectively by rapid transfer of the EGFP gene from a Donor Vector These recombinant vectors were then used to make virus using our BD BacPAK Baculoviral Expression System K1601 1 Panel A Shown above are Sf21 cells infected with recombinant virus pLP BacPAK9 EGFP Panel B pLP BacPAK9 6xHN EGFP kDa 198 115 93 fii Ii 50 36 29 Figure 3 Purification of 6xHN tagged EGFP from baculovirus using BD TALON Resin Lane 1 markers Lane 2 soluble lysate from Sf21 cells Lane 3 flowthrough Lane 4 wash with 5 mM imidazole Lane 5 elution with 150 mM imida zole Lysis wash and elution buffers all contain 20 mM Tris pH 8 0 and 100 mM NaCl The theo retical MW of 6XHN EGFP is 31 2 kDa AcM NPV sequences flanking the loxP site promote recombination with bac uloviral DNA to transfer the expression cassette to the polyhedrin locus of the baculoviral genome The BD BacPAK Baculoviral Expression Syste
21. are the property of Becton Dickinson and Company 2002 BD 2 New Products BD Atlas Plastic Human 12K Microarray The only calibrated microarray on the market A Calibration Procedure for Gene X e Rely on thoroughly tested long oligost for optimal specificity and minimal cross hybridization e Use a microarray that provides greater sensitivity e Our lot specific Calibration Standards provide more accurate data analysis Introducing the most powerful expression profiling tool to date from BD Biosciences Clontech the BD Atlas Plastic H uman 12K Microarray This array contains sequences from nearly 12 000 genes printed in duplicate on a plastic support surface The unique combination of high throughput gene expression with a plastic format promotes experimental efficiency lower background and accurate analysis In addition we print thoroughly tested long oligos on each array which provides superior specificity and sensitivity All these features make the BD Atlas Plastic Human 12K M icroarray the best choice for your expression profiling needs Make accurate and direct comparisons As a unique added feature of our plastic microarrays we calculate a Calibration Standard for every gene represented on the array This means that you can directly compare the results of plastic micro arrays from different lots and different experiments with confidence With each new lot of microarrays printed several microar
22. coding sequence for each reporter has been human codon optimized for efficient translation in mammalian cells Whether viewed by fluorescence microscopy Figure 1 or measured by flow cytome try these reporters emit at distinctive wavelengths that are easily resolved from our cyan green and yellow fluorescent variants So why not take your experi ments one step further and combine reporters to monitor two or even three different events simultaneously The tools are now at hand MCS Fluorescent protein pDsRed vaol Hs TK Express DR polyA polyA HcRed1 DR 4 2 kb E Kan Neo Poyao SV40 e orl Figure 2 Plasmid map for pHcRed1 DR and pDsRed Express DR Attention Drug Discovery Customers Are you interested in using BD Living Colors Fluorescent Proteins for your internal research BD Biosciences Clontech offers flexible research and drug discovery licenses that provide access to a complete set of novel Reef Coral Fluorescent Proteins RCFPs AmCyan Figure 3 BD Living Colors Reef Coral Fluorescent Proteins under UV light From left to right AmCyan ZsGreen ZsYellow Dsred AsRed and HcRed ZsGreen ZsYellow DsRed AsRed and H cRed Figure 3 Six distinct proteins four brilliant colors cyan green yellow and three spectrally distinct reds available exclusively to pharmaceutical and biotech companies through the BD Living Colors Licensing Program To learn more about ho
23. com between individuals and therefore are not included on our arrays AS an added benefit we provide clinical information for samples represented on these arrays so you Can investigate possible correlations between expression and patient history Rely on accurate sample representation Each sample cDNA on these arrays was generated from BD Premium RNA which means the original starting material was pure and intact see pages 8 9 Furthermore the cDNA was synthesized and amplified using our patented BD SMART Switching Mechanism At the 5 end of the RNA Transcript tech nology which ensures that the amplified CDNA retains the original complexity and relative abundance of the tumor and Clontechniques October 2002 Cancer Profiling Array Il and Tissue Specific Cancer Profiling Arrays continues A NTNTNT CE E EE g CE E EEE ft EEE ss p ee saa tt amp E pc ie amp amp tee ee TE TEES tin es Cell Lines _ e amp Controls _y B NTNTNT E m a E E 1 E E E E 7 1 E E a i E H z EF E T E Cell Lines amp Controls _y gt Figure 2 Differential gene expression on the Breast Cancer Profiling Array The Breast Profiling Arrays were hybridized using a radiolabeled probe for B actin Panel A or gelsolin Panel B Hybridization sig nals were detected by phosphorimaging Thirty pairs of BD SMART amplified CDNAs generated from b
24. enol chloro form extraction The M ulti 8 format comes with strips of 8 purification columns Notice to Purchaser NucleoSpin products are offered by BD Biosciences that allow you to process N X 8 samples Sle through a Hated shel ae Bea aD Aa mbH Inc a major manufacturer of products for analyti at once Figure 1 The M ulti 96 format Ba This partnership allows et provide the j igh quality products that you ve come to expect from includes 96 column plates that allow you a a BD Biosciences Clontech to meet your deicadd purifi to process n x 06 Samples at once j cation needs MACHEREY NAGEL s strict adherence to quality control standards maintains the reliability and performance of these purification products NucleoSpin Starter Kits Both the M ulti 8 kits are offered in a starter kit format which includes addi tional components required when using these kits for the first time A specially designed Tube Rack is included for hold ing the 8 column strips in the centrifuge Also Dummy Strips are provided for bal ancing and stabilizing the strips when fewer than 96 samples are being purified These components can be re used with Figure 1 NucleoSpin Multi 8 Blood purification non starter kits procedure Elution Pure genomic DNA Quick and easy protocols The N ucleoSpin protocols are designed to streamline the DNA purification process Once the biological fluids are loaded the purification usual
25. entical cultures grown in the presence of 10 of other lots of antibiotic free FBS In BD Tet Off cell lines gene expression is normally maximal in the absence of Tetracyclines Tc Tc levels are high enough in some lots of commercial serum to completely shut off TRE regulated genesina BD Tet Off Cell Line and to fully induce TRE reg ulated genes in a BD Tet On cell line data not shown RLU relative light units We now offer two different versions of Tet System Approved FBS The US Sourced FBS is the same high quality product that we have been supplying for years The new USDA Approved FBS undergoes all the same testing as US Sourced FBS is collected in USDA approved facilities and meets USDA stan dards for quality Both of our Tet System approved FBS types are now available in a trial size See for yourself the difference functional testing can make New Products Product Size Cat Tet Approved FBS US Sourced 50 ml 8630 y 500 ml 8630 1 Tet Approved FBS USDA Approved Q 50 ml 8637 y 500 ml 8637 1 Related Products BD Tet On System K 1621 1 BD Tet Off System K 1620 1 e BD RevTet On System K 1627 1 e BD RevTet Off System K 1626 1 e BD Adeno X Tet On System K 1652 1 e BD Adeno X Tet Off System K 1651 1 Coming Soon from BD Biosciences Clontech For more information on these products visit www clontech com Profile the effects of cancer treatments
26. esearch and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a com plete license These rights under the up front fee component may be purchased from Perkin Elmer or obtained by purchasing an authorized thermal cycler No right to perform or offer commercial services of any kind using PCR including without limitation reporting the results of purchaser s activity for a fee or other commercial consideration is hereby granted by implication or estoppel Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at the Perkin Elmer Corporation 850 Lincoln Centre Drive Foster City CA 94404 or Roche M olecular Systems Inc 1145 Atlantic Avenue Alameda CA 94501 This product is sold under licensing arrangements with F Hoffmann La Roche Ltd Roche M olecular Systems Inc and The Perkin Elmer Corporation Trademarks Axioskop is a registered trademark of Carl Zeiss Inc Gateway is a trademark and TOPO is a registered trademark of Invitrogen Corporation GeneSpring is a registered trademark of Silicon Genetics N ucleoSpin is a registered trademark of M acherey N agel GmbH amp Co Vent is a registered trademark of N ew England Biolabs BD BD logo and all other trademarks
27. genomic DNA content Genomic DNA contributes to a false representation of BD Biosciences Clontech www bdbiosciences com gene expression within a given RNA Sample and as shown here this type of contamination has a strong bearing on expression studies involving PCR By extension genomic DNA contamination would affect quantitative applications such as CDNA probe synthesis for microarrays and real time PCR yielding higher background and inaccurate results Our data here show that of the three commercially available sources of total RNA BD Premium Total RNA consists of intact RNA that is virtually free of genomic DNA resulting in a more accurate representation of gene expression using RT PCR Product Size Cat Price BD Premium Human Total RNA 5 O ug many 250 ug many BD Premium Mouse Total RNA 250 ug many BD Premium Rat Total RNA 50 ug many 250 ug many Also Available BD Premium Reserve RNA Our Premium Reserve RNA samples are isolated from extremely rare or difficult to obtain tissues and are available for custom packaging M ore than 100 human RN As are available and we offer both Premium Total and Poly ATRNAs In addition our collection features a number of matched tumor and normal RN As from individual patients Because quantities are so limited we are unable to offer these Premium Reserve RN As through our regular catalog For Premium Reserve RNA selection and ordering details contact
28. inding DNA consensus sequence 16 BD Biosciences Clontech www bdbiosciences com BD Mercury TransFactor Profiling Kits Size Cat Profiling Kit Oncogenesis 1 96 rxns K2073 1 Profiling Kit Oncogenesis 2 96 rxns K2075 1 Profiling Kit Oncogenesis 3 96 rxns K2076 1 Profiling Kit Inflammation 1 96 rxns K2062 1 Profiling Kit Inflammation 2 96 rxns K2072 1 BD Mercury Individual TransFactor Kits Size Cat NF B p50 Kit 96rxns K2058 1 STATI Kit 96 rxns K2059 1 cJun Kit 96 rxns K2061 1 cFos Kit 96 rxns K2065 1 CREB 1 Kit 96 rxns K2066 1 NFkB p65 Kit 96 rxns K2067 1 Rb Kit 96 rxns K2068 1 DP 1 Kit 96 rxns K2069 1 Related Product e TransFactor Extraction Kit K 2064 1 References 1 BD Mercury TransFactor Kits January 2002 Clontechniques X V1I 1 8 9 2 Shen Z et al 2002 Biotechniques 32 1168 1177 3 Two New BD Mercury TransF actor Profiling Kits April 2002 Clontechniques X VI1 2 20 4 BD Mercury TransFactor Profiling K it O ncogenesis 2 July 2002 Clontechniques XVII 3 15 t Patent Pending Table I Transcription factors profiled by BD Mercury TransFactor Kits Oncogenesis 1 DP 1 E2F 1 Rb p107 E2F 2 Sp 1 Oncogenesis 2 c Myb cMyc Max USF1 USF2 p53 Oncogenesis 3 HIF 1 HIF 18 Egr 1 CEBP Oct I Oct Il Inflammation 1 NF B p50 NF B p65 c Rel ATF2 CREB 1 c Fos Inflammation 2 c Jun c Fos FosB J
29. ins such as DsRed Express DR and HcRed1 DR eliminate the need for intense high energy radiation that may damage cells and tissues and their long wavelength emis sions stand out sharply against the green autofluorescent background from media culture ware and cellular components For detailed information about the spectral properties of HcRed1 and DsR ed Express please see References 5 and 6 In some kinetic assays rapid activation is as important as rapid inactivation Our data Figure 1 show that these new Clontechniques October 2002 New Products Destabilized DsRed Express and HcRed Vectors continued transcription reporters develop fluorescence soon after induction as expected from past studies of DsRed Express and H cRed1 the parent proteins whose maturation rates compare favorably to that of enhanced green fluorescent protein EGFP 5 6 Similarly when the inducer is withdrawn the fluorescence quickly declines due to the rapid turnover of the reporter Figure 1 Many destabilized vectors to choose from cyan green yellow and now red pDsRed Express DR and pHcRed1 DR Figure 2 join a growing line of BD Living Colors cyan green and yellow fluorescent vectors Like our other promoterless vectors pD sR ed Express DR and pHcRed1 DR contain an upstream multiple cloning site so that you can join any promoter enhancer element to the red reporter of your choice DsRed Express DR or HcRed1 DR The
30. iosciences Clontech www bdbiosciences com Clontechniques October 2002 17 BD Biosciences Clontech Discovery Labware Immunocytometry Systems Pharmingen BD Asia Pacific BD Singapore Tel 65 6861 0633 Fax 65 6860 1590 Japan Clontech Company Tel 81 3 5324 9609 Fax 81 3 5324 9636 Canada BD Biosciences Tel 888 259 0187 Fax 888 229 9918 Europe Belgium Tel 32 53 720 211 Fax 32 53 720 450 Latin America Caribbean Tel 55 11 5185 9995 United States BD Biosciences Clontech Tel 877 232 8995 option 4 Fax 650 424 0133 Discovery Labware Fax 978 901 7493 Immunocytometry Systems Fax 408 954 2347 Pharmingen Fax 858 812 888 Customer Technical Service Tel 650 424 8222 tech clontech com ww w bdbiosciences com INTERNATIONAL BD OFFICES amp DISTRIBUTORS ARGENTINA BD Argentina S R L Tel 54 11 4551 7100 x 106 Fax 54 11 4551 7400 AUSTRALIA BD Australia PTY Ltd Tel 02 8875 7000 Fax 02 8875 7200 AUSTRIA BD Biosciences Austria Tel 43 1 310 6688 Fax 43 1 310 7744 BELGIUM BD Biosciences Benelux N V Belgium Tel 32 5372 0550 Fax 32 5372 0549 BRAZIL BD Brazil Tel 55 11 5185 9995 Fax 55 11 5185 9895 CANADA BD Biosciences Tel 888 259 0187 Fax 888 229 9918 CENTRAL AMERICA amp THE CARIBBEAN BD CCA Tel 506 290 7318 Fax 506 290 7331 CHILE BD de Chile Tel 011 56 2 460 0380 x 21 Fax 011 56 2 460 0306 CHINA BD Asia Ltd China Tel 86 10 6418
31. l B In this case array calibration shows an insignificant difference in gene expression BD Biosciences Clontech www bdbiosciences com By eliminating false positives generated by noncalibrated arrays you save time for further study of real expression differences Depend on superior sensitivity Of course even a calibrated array is not an accurate tool if the printed oligos aren t reliable That s why we rigorously develop and test our oligo sequences to ensure optimal hybridization and sensitiv ity To accomplish this we develop a long oligo for each gene on the array Each long oligo is an 80 base DNA fragment Clontechniques October 2002 New Products BD Atlas Plastic Human 12K Microarray continued ee Te ee ad oe ee A E g H y Lid aor i m 7 es z iw Pa m e H E E E Moa SEA E a aT i ia m te ar 2 as ea E T z A a rf 7 nr i m BE g i i 7 iat oe ees ees LS ee oe SE fe T a 5 am a Tr sk E a ee Ja ee a aE Tr mc E a mm e E i _ ee i j y m i F 3 qa io rE _ i m z a m m am g a sag PLEN pan E a mi i ih m y ii a m m E em wi o a T m E m EL r ha _ i mi i ie am E 3 E a E E __ CE paT em a F a ee E p T m a ia i E a Be s W teami r a Figure 2
32. l intensities across the majority of genes BD Biosciences Clontech Mouse Universal Reference Total RNA Vendor S Mouse Universal Reference Total RNA Mouse Brain tissue Figure 1 Mouse Universal Reference Total RNA demonstrates more than 90 gene coverage We generated Cy 3 labeled probes using our Reference Total RNA another vendor s reference total RNA and RNA from mouse brain tissue Probes were hybridized to BD Atlas Glass Mouse 3 8 Microarrays 7907 1 We analyzed the expression results using GeneSpring Software version 3 2 2 to cluster genes according to their expression patterns A gene is considered expressed when its measured raw intensity is greater than or equal to 100 The red and blue colors indicate high and low expression respectively Varying shades of purple indicate the ratio of the intensity of any gene on each array to its median intensity across all arrays As shown here nearly all of the expressed genes from the mouse brain tissue were detected using our Reference Total RNA Furthermore among the genes detected with the Reference Total RNA 85 had intensities greater than or equal to the intensity obtained with the hybridization of any single tissue used to prepare our Reference Total RNA data not shown Our results indicate that the Reference Total RNA has more than 90 gene coverage with even distribution and outperforms another vendor s RNA mixture BD Biosciences Clontech e www bdbiosciences com
33. le Sp spleen Lane A 1 pg genomic DNA Lane B 10 pg genomic DNA Lane C 100 pg genomic DNA Lane D 1 000 pg genomic DNA tance to ascertain the extent of genomic DNA contamination in the RNA start ing material In this study we demonstrate the signifi cance of genomic DNA analysis in determining RNA quality by comparing BD Premium Total RNA with two other commercially available sources of total RNA Assaying for RNA quality As a first step we performed a routine RNA integrity test Figure 1 All samples are fairly uniform in appearance on a denaturing formaldehyde agarose EtBr gel producing bright 28S and 18S rRNA bands and an even smear between 0 5 and 11 kb Based on this assay alone all RNA samples contain intact RNA and thus appear to be equal in quality BD Biosciences Clontech www bdbiosciences com We then investigated differences in these total RN A samples by testing for genomic DNA A simple method for detecting genomic DNA contamination in RNA Samples is to perform a standard PCR using primers designed to amplify an intronic gene region Parallel comparison of PCR products generated from each RNA sample with those generated from serial dilutions of genomic DNA allows the estimation of genomic DNA present in RNA Figure 2 Results indicate that total RNA samples from two other commercial sources show considerable amounts of genomic DNA In contrast no genomic DNA contamination could be detected in BD
34. lity gel shift assay EM SA Today however many are discovering that DN A protein interactions can be measured with greater sensitivity and in shorter time using non radioactive 96 well plate assays developed by BD Biosciences Clontech 1 2 BD Mercury TransFactor Profiling Kitst are the new high throughput alternative to EM SA and supershift assays These kits provide a highly specific immunoassay for detecting and quantifying the DNA binding of several transcription factors involved in inflammation and oncogenesis 3 4 Our newest kit O ncogenesis 3 lets you measure HIF 1a HIF 18 Egr 1 c EBP Oct I and Oct Il adding six more entries to the long list of factors you can now profile with our ready to use kits Table 1 TransF actor Profiling Kits are ideal for studying transcriptional regulation in different cell lines and tissues and for investigating potential drug targets Each kit contains a 96 well plate Figure 1 for measuring the DNA binding behavior of six different transcription factors Individual wells have been precoated with the DNA consensus binding sequence for a specific factor To perform an assay add nuclear extract from mammalian cells to the wells and incubate to allow the transcription factor to bind its sequence Wash away the unbound proteins and add primary antibody Then add HRP conjugated secondary antibody incubate with HRP substrate and measure the color intensity Figure 1
35. ly takes less than 30 minutes depending on your centrifuge BD Biosciences Clontech www bdbiosciences com Clontechniques October 2002 13 New Products Destabilized DsRed Express and HcRed Vectors Red and far red fluorescent proteins engineered for rapid turnover Detect transient changes in gene expression e Develop stable cell lines e Monitor multiple events simultane ously choose from destabilized cyan green yellow and red fluorescent proteins Our newest BD Living Colors vectors pD sR ed Express DR and pH cRed1 DR encode destabilized variants of our red and far red fluorescent proteins D sR ed Express and HcRed1l In contrast to the original proteins these destabilized variants DsRed Express DR and HcRed1 DR have short half lives making them well suited for studies that require rapid reporter turnover These new promoterless vectors can be used to accurately analyze cis acting regulatory elements in studies of gene regulation and may facilitate routine generation of stable transfectants DsRed Express DR and HcRed1 DR were constructed by fusing the fluorescent pro teins to amino acid residues 422 461 of mouse ornithine decarboxylase M ODC one of the most short lived proteins in mammalian cells 1 This C terminal region of MODC contains a PEST sequence that targets the protein for degradation resulting in rapid protein turnover 1 2 Many potential applications Because of thei
36. m has special features that promote high recombination efficiency as well as high yields of protein for a eukaryotic system See inset Easy purification of 6xHN tagged proteins with BD TALON Resins You can express a protein bearing a 6xHN tag with pLP BacPAK 9 6xHN Once this protein is expressed it can be easily purified using BD TALON Resin our patented cobalt based immobilized metal affinity resin Figure 3 12 BD Biosciences Clontech www bdbiosciences com Product Size Cat pLP BacPAK9 Acceptor Vector 20 ug 6211 1 pLP BacPAK9 6xHN Acceptor Vector 20 ug 6212 1 Related Products BD BacPAK Baculovirus Expression System K 1601 1 BD BacPAK Baculovirus Rapid Titer Kit K 1599 1 BD Creator pDNR Cloning Kit K 1670 1 Cre Recombinase 8480 1 BD TALON M etal Affinity Resin 8901 BD TALON Superflow Resin 8908 BD TALON spin Columns 8902 BD TALON CellThru Resin 8910 BD TALON CellThru Disposable Columns 8914 BD TALON Purification Kit 1253 1 BD TALON 2 ml Disposable Gravity Columns K 8903 1 BD TALON Buffer Kit K 1252 1 Reference 1 Sauer B 1994 Curr O pin Biotechnol 5 521 527 BD BacPAK Expression System Features e High yield compared to mammalian expression systems e Greater similarity to naturally occurring protein due to the eukaryotic folding conditions e High recombination efficiency due to
37. mportant factor in determining RNA quality is the degree of genomic DNA contamination Genomic DNA is particularly trouble some in gene expression studies using PCR because its presence in total RNA Samples can generate positive expression data that does not truly represent actual expression levels in the RNA sample To validate expression data in these types of experiments it is of the utmost impor BD Premium Total RNA kb M CSM Sp Et SS ANO ARR BU 0 24 Vendor A Total RNA Ic SM Sp C SM Sp Vendor S Total RNA m a E A E Figure 1 Total RNAs from three commercially available sources appear uniform in RNA integrity For each sample 1 ug of human total RNA was heated to 37 C for 2 hr then subsequently analyzed using a denaturing formaldehyde agarose EtBr gel C colon SM skeletal muscle Sp spleen M 0 24 9 5 kb RNA ladder Invitrogen 15620 016 Vendor A Total RNA BD Premium Total RNA Vendor S Total RNA cC M l ice mMm lice M S A B C D Figure 2 BD Premium Total RNA is free of genomic DNA 1 ug of each human total RNA sample was used directly as a template for PCR using primers that amplify an intronic region of the MHC gene For human genomic DNA samples serial dilutions consisting of 1 10 100 and 1 000 pg were used as tem plates for PCR using the same primer set Products were analyzed using agarose EtBr gel electrophore sis C colon SM skeletal musc
38. n dancies among two or more lists and BLAST sequences against our database of probes The results displayed in text and table formats can be pasted into the Virtual Array Builder or any other application These on line resources the Gene List Separator Probe Finder Venn Operations and BLAST Probe Finder not only simplify your lists they also identify the best probes for detecting the gene s of interest The tools are free of charge and require no registration See our catalog for a full comparison of the nylon glass and plastic formats To find out more about our BD Atlas Array products and custom services log on to www clontech com atlas Clontechniques October 2002 New Products Mouse Universal Reference Total RNA Control RNA for improved microarray standardization e Rely on the broadest possible gene representation with minimal lot to lot variation Higher overall gene expression with a control made from various whole tissue sources e Use with any array or labeling method Comparisons of your microarray data just got easier with M ouse U niversal Reference Total RNA Our Reference Total RNA is made by pooling the total RNA extracts from a collection of different tissues yielding a mixture with the broadest possible gene representation available In addition our Reference Total RNA is produced on an industrial scale which minimizes variation between lots Our Reference Total RNA provides
39. n tifying cancer specific expression changes elucidating tumorigenic pathways or recognizing potential drug targets You can also focus your gene expression study on 30 samples of a specific tumor type Our Tissue Specific Cancer Profiling Arrays provide the benefits of the Cancer Profiling Array II for specific tissue types making focused expression profiling of your target gene easy and accurate T hese arrays are ideal for researchers studying breast colon or lung cancer or for researchers who suspect their target genes are associated with these types of cancer Since these arrays are manufactured on nylon membranes affixed to glass slides you can perform high throughput parallel hybridizations using a minimal amount of a standard radiolabeled probe and then easily obtain data from multiple slides using normal phosphorimaging techniques Acquire tissue diversity at a low price Eliminate the added time and expense of tissue acquisition RNA isolation and A 1 2 NT NI pa sa ia a NT l j i B 3 4 5 NT NT NT me EE f it Ea z ae Bi i EO 2 a E S 5 EE FE a ib a EE i EE ihe 12 13 14 1 haia B i E p E EE if rE be z p mi m ii 7 8 9 NT NT ii e Ubi cc tne i Oo N Xe E EFt shih Z zZ 4 ei se Ubi eae t i ot TT f ig 19 a n ah 15 16 17 we m E E E E a ms O pD cc ni Es k Ubi Figure 1 The Cancer Profiling Array Il demonstrates
40. n data using calibrated BD Atlas Plastic Microarrays Panel A After printing each lot of BD Atlas Plastic Microarrays sample arrays from the beginning middle and end of the printing run are hybridized with a mix of synthetic 33P labeled antisense oligonucleotides corre sponding to all genes on the array Then the intensity of each hybridization signal is quantitated by phosphorimaging and averaged Average antisense intensities are calculated for each gene as shown above for hypothetical Gene X Calibration Standards are then calculated for each array lot relative to the initial printing run All genes in the first printed lot Lot 1 as shown are assigned a Calibration Standard of 1 0 Panel B After normalizing arrays based on the overall signal intensities from all genes on the array experimental intensities for Gene X can then be compared using calculations that correct for array printing variations between lots Without this correction gene expression comparisons are less accurate and less reliable calculation of lot specific Calibration Standards for a target gene Then two different RNA samples are analyzed for target gene expression differences using two arrays one from each lot Panel B Without calibration the target gene appears upregulated Raw Signal Panel B Our practice of gene standardization demonstrates how the lot specific value corrects for typical printing variations across lots Calibrated Signal Pane
41. nual PT 3578 1 Related Products Matched Tumor N ormal Expression Array 7840 1 e Cancer Profiling Array 7841 1 Autoimmune Disease Profiling Array 7843 1 e Blood Disease Profiling Array 7842 1 References 1 Zhumabayeva B et al 2001 BioTechniques 30 1 158 63 2 Zhumabayeva B et al July 2000 Clontechniques X V 3 22 23 Sers C et al 2002 O ncogene 21 2829 2839 4 Wiechen K et al 2001 Am J Pathol 159 1635 1643 o Notice to Purchaser BD SMART technology is covered by U S Patents 5 962 271 amp 5 962 272 The PCR process is covered by patents owned by Hoffmann LaRoche Inc and F Hoffmann LaRoche Ltd Table Patient representation for each tissue type included on Cancer Profiling Array Il Number of samples Tissue type Breast ovary colon stomach lung kidney uterus cervix rectum thyroid testis skin 10 Small intestine pancreas Bladder vulva Prostate Trachea liver w A UN Clontechniques October 2002 7 Technical Note BD Premium Total RNA Contains Virtually No Genomic DNA an Important Factor in RNA Quality Jim Yan Bakhyt Zhumabayeva Ph D and Michael Herrler Ph D Gene Cloning and Analysis Group BD Biosciences Clontech In this study we performed RT PCR and PCR using commercially available total RN A samples and BD Premium Total RNA We found that for expression applications requiring PCR RNA integrit
42. on vector provides easy purification with BD TALON Resins Do you need to express your protein ina baculoviral system use vectors that elimi nate complicated subcloning procedures and let you proceed directly to expression in the shortest time possible Our new pLP BacPAK9 and pLP BacPAK 9 6xH N Vectors do just that These BacPAK 9 Shuttle Vectors are BD Creator Acceptor Vectors that provide efficient subcloning and compatibility with Baculoviral expression systems like our BD BacPAK Baculovirus Expression System K 1601 1 and BD Biosciences Pharmingen s Baculo Gold Expression System BD Creator technology ensures high efficiency cloning These vectors act as BD Creator Acceptor Vectors because they contain the loxP sequence from the P1 bacteriophage 1 instead of a multiple cloning site In BD Creator cloning Cre Recombinase transfers a gene of interest from any BD Creator Donor Vector into any BD Creator Acceptor Vector in just 15 minutes without restriction digestion or ligation 1 This method of subcloning is extremely efficient Figure 1 Quickly focus on protein expression After transferring your gene of interest to the expression cassette of the shuttle vector you can express the protein as part of the Baculoviral genome Figure 2 The ko 123 45 67 89 1011 Figure 1 Efficient BD Creator transfer of EGFP gene from Donor Vector to pLP BacPAK9 6xHN Acceptor Vector Lane 1 1 kb molecul
43. or clone one for you After you select the desired genes and enter any of your own tell us which BD Atlas Array to print nylon glass or plastic our newest support designed especially for high density print ing Like glass plastic arrays provide a rigid non porous surface that resists non specific binding but like nylon they can be analyzed by phosphorimaging Finally tell us how many arrays you would like printed The price is instantly calculated Arrays printed in the format of your choice BD Atlas Plastic BD Atlas Glass BD Atlas Nylon Figure 1 Designing BD Atlas Custom Arrays on line This simple flow chart illustrates the process for designing custom arrays If you are starting with your own list of genes in the form of GenBank LocusLink or Unigene Accession numbers or DNA sequences you may sort the list using one of our bioinformatics tools With Venn Operations you can even compare two lists to produce a single non redundant list Next use our Gene Search and Virtual Array Builder to compile a list of unique sequences for custom printing Finally choose the desired surface nylon glass or plastic To order the array submit your request on line A written confirmation of your order will be sent by e mail BD Biosciences Clontech www bdbiosciences com Product Size Cat Price BD Atlas Custom Plastic Microarray each CS2050 1 inquire BD Atlas Custom Glass Microarray each
44. our cDNA or oligo microarray studies These genomic approaches recognize the expression dif ferences of many genes when comparing normal tissues with tumor tissues W hen you have identified candidate genes that are either up regulated or down regulated in tumors use our Cancer Profiling Arrays to further define these genes roles in particular tumor types and at particular tumor stages 3 4 Simply generate a radiolabeled probe for your gene of interest and hybridize it to your chosen array Using the Cancer Profiling Arrays in this way can serve as a vital step in the identi fication of potential cancer drug targets Because development is a costly endeavor swift validation of candidate genes is essential to focusing on promising thera pies With our Cancer Profiling Arrays you can generate statistically significant proof of a gene s relevance to cancer and to particular tumor types BD Biosciences Clontech e www bdbiosciences com New Products Product Size Cat Cancer Profiling Array II each 7847 1 Breast Cancer Profiling Array each 7844 1 Lung Cancer Profiling Array each 7845 1 Colon Cancer Profiling Array each 7846 1 Components Cancer Profiling Array Hybridization Chamber for Tissue Specific Cancer Profiling Arrays 2 Wash Containers for Tissue Specific Cancer Profiling Arrays Human Ubiquitin Control cDNA Probe BD ExpressH yo Hybridization Solution Orientation Grid User M a
45. r rapid turnover D sR ed Express DR and HcRed1 DR are useful as transcription reporters for measuring both the up and down regulation of pro moter activity For example by placing DsRed Express DR or HcRed DR under the transcriptional control of a cis acting regulatory element you can develop assays to study the induction and repres sion of gene expression during signal transduction Figure 1 Similar constructs could also be designed to explore the programmed changes that occur during embryogenesis and cell differentiation Such studies have been carried out with destabilized green fluorescent protein 3 4 And because the fluorescence can be detected without the addition of substrates or cofactors you can measure the events non invasively in real time a real advan tage over other transcriptional reporters such as luciferase or B galactosidase A 120 ia E 100 8 s830 60 Ee c g 40 20 pNF B DsRed Express DR B TNF D TNFa TNF a 4hr ei TNF o 4 hr TNF a 12 hr DNF KB HcRed1 DR TNF c Figure 1 Destabilized red fluorescent proteins measure both the up and down regulation of promoter activity Panel A To measure the activation of NFxB a transcription factor known to regulate several genes involved in inflammation immune response and apoptosis 7 8 the NF B DNA response ele ment was cloned into the MCS upstream of the fluorescent reporter gene in pDsRed Express DR and
46. rane and the spots are more uniform and discrete facilitating accu rate automated analysis The plastic support is rigid and resistant to warping at high wash temperatures so the array does not distort and complicate image analysis Combine these features with the easier improved automatic grid alignment featured in our new BD Atlaslmage 2 7 Software and you get a microarray that delivers quality data in little time The Plastic Human 12K M icroarray also furnishes you with a powerful option in data analysis Because the gene coordi nates represented on the BD Atlas Plastic Human 8K M Icroarray have been maintained on the 12K M icroarray you can easily calibrate your 12K M icroarray gene signals to the corresponding values generated on the 8K M Icroarray This allows you to further compare your data from 8K M icroarray experiments by adding new gene expression data With this feature you save time by building upon existing data instead of starting over BD Biosciences Clontech e www bdbiosciences com Product Cat BD Atlas Plastic Human 12K Microarray 2 arrays 7931 1 Size Components e 2 Plastic Human 12K M icroarrays BD PlasticH yb H ybridization Solution BD Atlas Nucleospin Extraction Kit dNTP M ix BD PowerScript Reverse Transcriptase BD PowerScript Reaction Buffer Random Primer M ix with Synth Control e DTT Termination M ix Human Placenta Control Poly ATRNA Deionized H O Gene List C
47. rays some from the begin ning middle and end of the printing are hybridized using an antisense oligo cali bration mixture Following quantitation the resulting lot specific calibration values are averaged and listed on our web site www clontech com atlas atlasimage T hese values are easily imported into BD Atlas mage Software which will automatically calculate standardized array signals yielding the most accurate and meaningful array comparisons This standardization protocol is ideal for data base generation as it allows statistically significant data to be generated from microarrays printed at different times Figure 1 describes the importance of array calibration to generate accurate mean ingful results Panel A first illustrates the Lot 1 Average Antisense Intensity 9 arrays Calculated 400 Calibration 400 Standard Calibrated to Lot 1 1 Lot 2 390 415 405 320 333 339 GIE H GIE H 386 411 378 307 330 343 I H IH 419 393 404 325 311 317 EGIA H EGIA H e a G U E 9 9 325 B Experimental Analysis of Gene X Sample A hybridized to array from Lot 1 Sample B hybridized to array from Lot 2 Sample A Sample B Expression Interpretation Lot 1 Signal Intensity Lot 2 Signal Intensity Ratio Raw Signal 500 350 390 1 43 Misleading No Calibration Calibrated 500x1 500 350x1 23 4305 200 1 16 Valid 430 5 Signal Intensity x Calibration Standard Figure 1 More accurate expressio
48. reast normal and tumor samples are spotted on the upper portion of the glass slide The array also includes three breast cancer cell line CDNAs MCF7 MDA MB 231 and MDA MB 435S Positive controls ubiquitin CDNA and negative controls yeast total RNA yeast tRNA E coli DNA poly A human Cot 1 DNA and human genomic DNA are spotted on the lower portion of the glass slide N normal T tumor normal RNA samples 1 2 High quality starting materials and accurate sample representation mean you can have complete confidence in your results Achieve accurate expression results All samples on these arrays are normalized to two different housekeeping genes B actin and ubiquitin This normalization process is an integral part of our array quality N ormalization ensures a consistent hybridization signal for all the samples represented on the array while also assuring you that a true differential expression pattern exists for your gene of interest You can be confident that your results are not due to variances in cDNA content between spots Figures 1A and 2A demonstrate the uniform quality of these arrays when probed with a consti tutively expressed housekeeping gene Using the candidate tumor suppressor gene gelsolin as a probe however distinguishes a differential expression pattern in specific tumor types Figures 1B and 2B Identify disease markers for drug discovery You can use our Cancer Profiling Arrays to complement y
49. sekeeping gene phospholipase A2 Figure 3 Panel A All samples generate the expected 700 bp fragment H owever a separate experiment in which all RNA samples were used directly as a template for a standard PCR omitting the prior RT step and Table I Products that use BD Premium RNA Gene Expression Analysis BD MTN Blots BD MTE Arrays BD MTC Panels Total RNA Panels Cancer and Disease Profiling Arrays Tumor Normal Matched cDNA Pairs and Panels Gene Cloning BD Premium Total RNAs and Poly At RNAs BD QUICK Clone cDNAs BD Marathon Ready cDNAs using the same primers as before also produces the same fragment for some samples thus showing evidence of genomic DNA contamination Figure 3 Panel B These results indicate that a portion of the product generated in the RT PCR is in fact due to the presence of genomic DNA in theRNA starting material for some samples Furthermore these results suggest that genomic DNA contributes to an inaccurate representa tion of this gene s expression N otably however BD Premium Total RNA demonstrates the least genomic DNA contamination among commercially available RNA tested in this study We conclude that RNA integrity testing is not sufficient to guarantee RNA quality Genomic DNA contamination is also a critically important parameter Although all RNA samples tested in this study are intact without RNA degrada tion these samples vary widely in their
50. t can be cloned with this kit The BD In Fusion PCR cloning method does not require the presence of A overhangs so you can use any thermostable poly merase for amplification including proof reading enzymes such as Vent and Pfu For PCR amplification we recommend our BD Advantage 2 Polymerase M ix 8430 1 a robust enzyme mix that is ideally suited for long distance LD PCR and has been thoroughly tested with the BD In Fusion protocol BD Creator System The BD In Fusion PCR Cloning Kit makes it easy to clone and characterize products Figure 2 After you obtain a cDNA of interest the BD Creator System enables directional single step fast and precise transfer of genes from oD NR Dual to any one of our Acceptor Vectors Then our wide variety of expression systems allow you to express the gene to study protein protein inter actions protein localization gene expression patterns gene function and more TOPO Directional TA cloning kits TOPO T A cloning A J 5 30 min 5 30 min Overnight Some kits A Some kits J J A Gateway None BD Fusion Blue Competent Cells High efficiency competent cells opti mized for use with cutting edge cloning and expression technologies e Oneshot transformation aliquots e PCR cloning e BD Creator recombination e Low background e High efficiency BD Biosciences Clontech www bdbiosciences com Product Size Cat BD In Fusion PCR Cloning Kit 50 rxns K1916 1
51. the design of the BacPAK6 Viral DNA Clontechniques October 2002 New Products New NucleoSpin Nucleic Acid Purification Kits Medium and high throughput DNA purification from Blood and Virus e Fast easy protocol completed in Samples e g 4 x 8 NucleoSpin Products Q under 30 minutes aie Cat F a e Medium and high throughput E rie ae K3096 y formats g 4 x 96 K3096 1 Lysis Multi 8 Virus Starter Kit No phenol chloroform extraction 12x8 K3097 1 Multi 8 Virus Kit l 7 K3097 2 Introducing three new kits for medium w eS j 2 en or high throughput nucleic acid purification O _ Multi 8 Blood Tissue Starter Kit Dummy Strips 12 x8 K3098 1 from blood and virus The N ucleoSpin M ulti 8 Blood Kits allow you to purify genomic DNA from 200 ul samples of whole blood plasma serum or other biological fluids The NucleoSpin M ulti 8 Virus Kits and NucleoSpin M ulti 96 Virus Kits allow you to purify viral RNA or DNA from 100 ul samples of plasma serum or cell free biological fluids Binding Multi 8 Blood Tissue Kit 60 x8 K3098 2 Related NucleoSpin Products e Blood M ini K 3052 1 2 paring e Blood M idi K 3054 1 e Blood XL K3095 1 Blood QuickPure K 3082 1 e M ulti 96 Blood K 3062 1 Washing e Virus K3055 1 e Virus M idi K 3061 1 These kits are designed for fast process ing of a flexible number of samples with out the inconvenience of ph
52. tissue specific expression of gelsolin The Cancer Profiling Array Il was hybridized separately with a radiolabeled probe for the housekeeping gene ubiquitin Panel A and a radiolabeled probe for gelsolin Panel B Hybridization signals were detected by phosphorimaging Numbers indicate tissue types in columns 1 breast 2 ovary 3 colon 4 stomach 5 lung 6 kidney 7 bladder 8 vulva 9 prostate 10 uterus 11 cervix 12 rectum 13 thyroid gland 14 testis 15 skin 16 small intestine 17 pancreas 18 trachea 19 liver N normal T tumor Ubi ubiquitin CDNA cc cancer cell line cDNAs membrane manufacture Our Cancer Profiling Arrays are the ideal choice for high throughput multiple tumor analysis With the Cancer Profiling Array II you can proceed directly to determining your target gene s expression in a variety of tissue types representing various stages of disease Alternatively choose a Tissue Specific Cancer Profiling Array to simul taneously survey the expression pattern of your gene in 30 different tumor samples and their corresponding normal tissues from individual patients Because each matched pair of cDNAs on these arrays comes from an individual patient you can be sure that any differential expression you see is due to actual differences between tumor and normal tissue Pooled samples from multiple patients can mask differences in gene expression patterns BD Biosciences Clontech www bdbiosciences
53. unD Sp 1 STAT1 Clontechniques October 2002 Tet System Approved FBS More options to achieve the best results e Functionally tested for optimal Tet induction e The only choice for Tet induced expression of toxic proteins e Two options US Sourced our premium FBS USDA Approved our economical alternative The problem Trace tetracycline contami nants in standard Fetal Bovine Serum FBS can alter experimental results by enabling background expression in BD Tet On Systems and suppressing maximum expression levels in BD Tet Off systems The solution Tet System Approved FBS from BD Biosciences Clontech the only functionally tested FBS approved for use with our BD Tet On and BD Tet Off Systems The functional testing makes our FBS superior because it ensures that you will be able to achieve the full range of inducibility and the lowest background possible with our Tet Systems Figure 1 Just testing for antibiotics in FBS may not detect trace levels of tetracycline and its derivatives Trace amounts can signifi cantly alter the inducibility of the Tet Expression Systems Some Tet cell lines can be affected by as little as picograms ml concentrations of tetracyclines E Our FBS Other FBS Luciferase Activity RLU x 10 Figure 1 Serum source affects luciferase expres sion levels in BD Tet Off Cells BD Tet Off Cells grown in the presence of 10 Tet System Approved FBS were compared to id
54. us on a Select set of genes Then why not design your own expression array using our BD Atlas Custom Array Printing Services Simply provide us with the GenBank LocusLink or BD Biosciences Clontech ID numbers of the genes you are interested in and we will print the array for you Affordable and flexible BD Atlas Custom Arrays are meticulously engineered to ensure accurate reliable and repro ducible results In fact genes exhibiting greater than a three fold change in expression using BD Atlas Arrays are confirmed by RT PCR with a frequency of over 90 Thus custom arrays are ideal for performing new experiments or for confirming results obtained with more Identify genes of interest Gene List Separator Probe finder Venn Operations Blast Probe Finder BioCalculator Design your Custom Array BD Atlas Custom Array order form extensive arrays that compare thousands of genes Design your array on line You can design your custom array on line through our Bioinformatics home page at bioinfo clontech com H ere our BD Atlas Gene Search amp Virtual Array Builder lets you choose from over 13 000 human 4 000 rat and 8 000 mouse genes as it guides you through the design and order process Figure 1 You can select from our extensive collection of cDNAs and long oligos or submit your own list of genes If we do not currently have an oligo or CDNA sequence that matches your gene we will synthesize
55. w these reporters can illuminate your research log on to the RCFP family home page at www clontech com products families RC FP W hile there be sure to download a free copy of the BD Living Colors Licensing Program brochure which describes all six RCFPs in vivid detail To reach us directly call our Licensing H otline at 800 662 2566 extension 7816 outside the U S contact your local BD Biosciences representative or email us at licensing clontech com BD Biosciences Clontech www bdbiosciences com Product Size Cat pDsRed Express DR Vector 20 ug 6996 1 pHcRed1 DR Vector 20 ug 8114 1 References 1 Li X amp al 1998 J Biol Chem 273 34970 34975 2 Rechsteiner M 1990 Sem Cell Biol 1 433 440 3 Wahlers A et al 2001 Gene Ther 8 477 486 4 Dorsky R I amp al 2002 Dev Biol 241 229 237 5 BD Living Colors HcRed April 2002 Clontechniques X VII 2 12 13 6 BD Living Colors DsRed Express J uly 2002 Clontechniques X VIII 3 16 17 7 Jesenberger V amp Jentsch S 2002 N ature Reviews 3 112 121 8 Baldwin A S 1996 Annu Rev Immunol 14 649 681 Notice to Purchaser of DsRed and HcRed Products Not For Profit Entities Orders may be placed in the normal manner by contacting your local representative or BD Biosciences Clontech Customer Service at either 800 662 2566 or 650 424 8222 extension 1 BD Biosciences Clontech grants not for profit research entities
56. y alone is not sufficient to generate accurate results The degree of genomic DNA contamination in an RNA sample is an equally important determinant in generating quality data O ur results also show that BD Premium Total RNA contains intact RNA with virtually no genomic DNA BD Premium Total RN As are high quality RNAs useful in a variety of appli cations including library construction BD Atlas Array hybridizations RT PCR analysis CDNA synthesis N orthern blotting and RNase protection assays RPAs Each Total RNA sample is prepared using a modified guanidinium thiocyanate method and rigorous quality control tests confirm that each preparation consists of intact full length RNA with virtually no genomic DNA Determination of RNA quality is essential to any application that utilizes RNA RNA quality is usually confirmed by the electrophoresis of a 0 5 1 ug RNA sample on a denaturing formaldehyde agarose EtBr gel to check for integrity Human total RNA samples should produce an even smear between 0 5 and 12 kb with two bright 28S and 18S rRNA bands at approximately 4 5 and 1 9 kb respectively The ratio of the intensities of 28S to 18S rRNA bands should be at least 2 1 A decrease in the intensity ratio to 1 1 or a downward shift in the RNA smear indicates degraded RNA Poor quality RNA can lead to high background or inaccurate expression results due to the absence of intact full length RNA H owever another i
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