Antibody validation with VERIFY Antigen Standards, a newly released line of overexpressed

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1 GeneDex 2008 Volume 3 Issue 4 Editor: Michele Nealen Also In this Issue: Three ORF vectors you shouldn t be without! Page 7 Exact-shRNA, the new custom shrna service, is here! Page 19 RNAi and the influenza pandemic Page 21 Antibody validation with VERIFY Antigen Standards, a newly released line of overexpressed antigen lysates by Donghui Ma, Ph.D., OriGene Technologies Inc. Antibodies are among the most straightforward and useful tools to detect protein expression. Currently, there are thousands of antibodies available on the market, which may cover more than half of the human proteome. However, life science researchers know that it can be like searching for a needle in a haystack to find one reliable, functional antibody against their favorite gene. Huge amounts of time, effort, and research dollars can be spent to fully characterize a single antibody at one s own lab. OriGene, the leader in the cdna cloning market, is now offering more than 10,000 tagged overexpression lysates, as the VERIFY Antigen Standard line. VERIFY Antigen Standards are 100 ug of whole cell lysates containing a tagged, overexpressed protein of your choice. These lysates are the ideal reagent to validate Continued on Page GeneDex_V3_4_12-8.indd 1 12/9/08 11:55:07 AM

2 Continued from Page 1 an antibody prior to an experiment, or confirm expression when a protein-specific antibody doesn t exist. Each VERIFY lysate contains a full-length expressed human protein with a Myc and a DDK** tag for use in Western blot experiments and other applications. Each kit also includes a vector-only transfection lysate as a negative control. The transiently overexpressed cell lysates have been validated in Western blots with a mouse monoclonal anti-myc antibody (TA100010) or an anti-ddk antibody (TA100011) (Figure 1). Figure 1. MW kda Anti-Myc antibody SRY P14K2B POU5F1 PMAIP1* UCHL3 ACVL1 LGR5 TLR9 PRKCE CLIC1 TGFB2 OriGene s VERIFY Antigen Standards can be used: to qualitatively evaluate the difference between commercially available antibodies to compare batch-to-batch differences for an antibody, especially for rabbit polyclonal antibodies to quantify an endogenous antigen concentration as Western blot positive controls to investigate protein-protein interactions in protein microarrays to decode antibody reactivity for immunoprecipitation MW kda SRY P14K2B POU5F1 Anti-DDK antibody PMAIP1* UCHL3 ACVL1 *molecular weight is smaller than 10kDa. LGR5 TLR9 PRKCE CLIC1 TGFB2 Figure 1. Western blot experiments with VERIFY Antigen Standards detected with anti-tag antibodies. The lysates were probed with an anti- Myc antibody (top panel) or anti-ddk antibody (bottom panel). Most proteins are easily detected and are visible as a single band. Multiple bands may be due post-translational modification of the protein, proteolytic degradation, etc. Still not sure that you need to validate your antibody? Consider the following experiments (Figure 2). VERIFY Antigen Standards were used to compare multiple commercial antibodies for two different targets. The experiments demonstrated an obvious difference in quality of antigen detection between the different sources of each antibody. Using VERIFY Antigen Standards for this pre-experiment analysis will save researchers significant time and resources, as it would otherwise require the production and use of relevant samples that can now be saved for the research experiments themselves. These data show that VERIFY Antigen Standards can easily and consistently compare the immunoreactivity of multiple antibodies, allowing us to determine the detection sensitivity and specificity for each GeneDex_V3_4_12-8.indd 2 12/9/08 11:55:08 AM

3 Figure 2. Figure A B C P53-myc-DDK 10ug/lane ng 10ng 5ng 1ng.5ng A431 HT-29 HEK293T 28 Antibody A ***** Antibody B *** Antibody C ** kda ng 45ng 22ng 4.5ng 2.2ng HEK293T kda ng 45ng 22ng 4.5ng 2.2ng HEK293T kda ng 45ng 22ng 4.5ng 2.2ng HEK293T Anti-p53 kda Antibody D * 221ng 45ng 22ng 4.5ng 2.2ng HEK293T kda Antibody E * 221ng 45ng Figure 2. Western blot experiments with VERIFY Antigen Standards identify differences in sensitivity and specificity between multiple sources of antibodies. Upper panel: Three commercial (protein-specific) antibodies were used to probe the VERIFY lysates for human STAT3 (NM_139276). Lanes A (probed with antibody A) show strong antigen binding, lanes C (antibody C) show weak binding, while lanes B (probed with antibody B) show no reactivity with the antigen. Middle and lower panels: Five different sources of antibodies were used to probe immunoblots containing Myc and DDK-tagged VERIFY lysates for human TP53 (NM_000546) that had been quantitated and serially diluted. Whole cell lysates from HEK293T cells (2 ug/lane) were included for comparison. Biological research sometimes demands quantitative protein measurements and OriGene s VERIFY Antigen Standards can facilitate this. Figure 3 is an example of OriGene s lysates used as a standard to quantitate endogenous TP53 protein levels in different carcinoma cell lines. 22ng 4.5ng 2.2ng HEK293T Figure 3. VERIFY Antigen Standards for human TP53 (NM_000546) were quantitated and serially diluted prior to loading next to 10 ug each of whole cell lysates from the cell lines A431, HT-29, and HEK293T. The blot was probed with a protein-specific antibody against TP53, and the immunoreactive bands were measured. Generation of a standard curve based on the intensity of the bands visualized for the VERIFY standards allows the estimation of the amount of TP53 protein expressed in the experimental cell lines. To generate the 10,000 currently available VERIFY Antigen Standards, the human HEK293T cell line was transiently transfected using MegaTran 1.0 Transfection Reagent (TT200002) and 5 ug of a TrueORF cdna plasmid (an overexpression construct in the pcmv6-entry vector; see com/orf/ and Figure 4 for more details). Transfected cells were cultured for 48 hours before harvesting. The cells were lysed in modified RIPA buffer (25 mm Tris-HCl ph 7.6, 150 mm NaCl, 1% NP-40, 1 mm EDTA, 1x Proteinase inhibitor cocktail mix (Sigma), 1 mm PMSF and 1 mm Na 3 VO 4 ), and then centrifuged to clarify the lysate. Protein concentration was measured by a BCA kit (Thermo Scientific Inc.). Cell lysates were aliquotted and lyophilized at -20ºC before shipping. Continued on Page GeneDex_V3_4_12-8.indd 3 12/9/08 11:55:09 AM

4 Continued from Page VERIFY Antigen Standards are 100 ug of whole cell lysates containing a tagged, overexpressed protein of your choice. Each VERIFY lysate contains a full-length expressed human protein with a Myc and a DDK** tag for use in Western blot experiments and other applications. Figure 4. Kan r / Neo r SV40 ori HSV TK Poly A f1 ori CMV promoter pcmv6-entry 4929 bp MCS myc tag DDK tag PolyA signal ColE1 Figure 4. Plasmid map of pcmv6-entry, the construct used to overexpress the tagged proteins in every VERIFY Antigen Standard. This plasmid is designed for high level overexpression of a full-length human cdna clone, tagged at the C-terminus with Myc and DDK. As the old saying goes, Trust, but verify. You can trust that your new antibody will work the first time, in the generic conditions specified. But why not VERIFY your antibody before using your valuable time and samples? VERIFY Antigen Standards are available on the web at lysate/, or you can place your order by fax ( ), to custsupport@origene.com, or by phone ( ; use outside the US). ** Peptide sequence of the DDK-tag (Flag ): N-DYKDDDDK-C. Flag is a registered trademark of Sigma-Aldrich. ORF T7 Promoter Sgf I Asc I Rsr II Mlu I Not I Xho I VERIFY Antigen Standards Protocols Sample Preparation, Electrophoresis and Protein Transferring: 1. VERIFY antigen standards are shipped as lyophilized lysate at ambient temperature. Add 100 ul of 1X SDS Sample Buffer (provided) to the vial containing 100 ug lysate, then aliquot the resuspended lysate (we suggest 5-10 ug per tube). Immediately freeze the unused aliquots to -20 C for long-term storage. 2. If reducing conditions are required, add 50 mm DTT (or betamercaptoethanol to a final concentration of 2.5%) into the sample tube. Boil the samples for 5 minutes*, then incubate on ice before loading. Prepare a protein standard (SeeBlue Plus 2 Prestained Standard, Invitrogen #LC5925) as well. We recommend using OriGene s MYC/DDK Tagged Western Blot Molecular Weight Markers (#MWM1001) if anti-tag antibodies will be used for probing. 3. Load all samples and standards into the wells of a pre-cast SDS polyacrylamide gel, filling unused wells with 1X SDS sample buffer. We recommend using NuPAGE Novex 4-12% Bis-Tris Mini Gel (Invitrogen #NP0323BOX) for proteins with molecular weights smaller than 200 kda. 4. Run the samples at 170V (constant voltage) for 40 to 60 minutes, or until the loading dye reaches the bottom of the gel. Remove the gel and soak in protein transfer buffer (see recipe on page 5) for 15 minutes. 5. Cut a nitrocellulose membrane (ISC BioExpress #F ) to the size of the area of the gel to be transferred. Prewet the membrane in transfer buffer. 6. Assemble the electroblotting cassette and place the electrodes in the blotting unit, according to the manufacturer s instructions. Fill the transfer unit with protein transfer buffer. 7. Perform the transfer at 100 V for 1 hour at constant current (not to exceed 0.4 A), preferably at 4 C. 8. Following transfer, remove the membrane from the blotting cassette and mark the orientation of the gel with a pencil. Rinse briefly with PBS. This membrane may be used for immediate antibody probing, or stored at 4 C, securely wrapped in plastic wrap. * If your VERIFY antigen standard is a multipass transmembrane protein, boiling can cause protein aggregation that will prevent appropriate migration in the acrylamide gel. In this situation, we recommend that you instead incubate the lysate in 1X SDS sample buffer at room temperature for 30 minutes prior to loading GeneDex_V3_4_12-8.indd 4 12/9/08 11:55:10 AM

5 protein transfer buffer Tris base 48.5 g Glycine 0.2 g Methanol 3.2 L 10N NaOH 1.5 ml add water to 16 L Antigen Detection: 1. Wash the membrane with TBST (see recipe below) once for 5 min at room temperature. Block non-specific binding on the membrane with freshly prepared 5% nonfat dried milk in TBST for 1 hour on a shaking platform at room temperature. 2. Incubate the membrane with a specific primary antibody diluted in 5% nonfat dried milk/ TBST at the manufacturer s recommended dilution with gentle agitation at 4 C overnight, or for several hours at room temperature. 3. Remove antibody solution, and wash the membrane three times for 5 min each with TBST. 4. Incubate the membrane with OriGene s HRP-conjugated goat anti-mouse secondary antibody (#TA100015, or other secondary antibody as appropriate) at 1:20,000 in 5% nonfat dried milk/ TBST for 1 hour at room temperature with gentle agitation. 5. Wash the membrane three times for 5 minutes each with TBST. 6. Prepare OriGene s Western Blot Luminol Reagent (# TA100016) according to specifications, mixing reagents A and B in equal volumes immediately prior to use. 7. Lay the membrane on a plastic surface with the transferred protein side up. Add the prepared luminol solution to the membrane, and incubate for 1 minute. Remove the excess solution and wrap the membrane well with transparent plastic wrap. 8. Place the wrapped blot (protein side up) in an X-ray film cassette. Place a sheet of X-ray autoradiography film on the top of the membrane. Close the cassette for 15 sec to 1 min. Remove the film for development. Add additional films if needed for longer or shorter exposures. TBST 10 mm Tris-HCl, ph mm NaCl 0.05% Tween 20 Frequently Asked Questions about VERIFY antigen standards What makes OriGene the optimal source for antigen standards? Answer: All of OriGene s overexpression cell lysates are prepared from HEK293T cells transfected with TrueORF cdna clones. Each expressed protein has a C-terminal Myc/DDK tag which can be monitored by anti-tag antibodies for expression verification. OriGene has the most comprehensive genome coverage represented by full-length human ORF clones. Do all TrueORF clones produce the same amount of overexpressed proteins? Answer: Each clone is different. It is difficult to predict or control specific protein expression levels. We provide VERIFY antigen standards as 100 ug of total protein lysate. Can I use these VERIFY antigen standards for applications other than Western blotting? Answer: The lysates are supplied in a lyophilized form, and based on our experience, it is difficult to reconstitute the lysate in a buffer that does not contain SDS. Therefore, you may not be able to use a resuspended lysate for any application other than SDS-PAGE. But these standards can potentially be used in many other applications, such as antibody production, immunoprecipitation studies, protein/protein interactions, etc. If you are interested in receiving your VERIFY antigen standard in another format (e.g., frozen in RIPA buffer), please contact OriGene Technical Support for custom options. What are the terms of OriGene s VERIFY antigen standard guarantee? Answer: We guarantee that our overexpression cell lysates pass through stringent Western blot testing using anti-tag antibodies. Each lysate is supplied with a Western blot image showing a positive protein band at or near the correct molecular weight. No guarantee is provided for assays other than Western blotting. Continued on Page GeneDex_V3_4_12-8.indd 5 12/9/08 11:55:10 AM

6 Continued from Page 5 How long can I store these lysates? Answer: The lysates can be stored either in the lyophilized form or after reconstitution in 1X SDS buffer. OriGene guarantees 12 month stability from date of shipment when these samples are properly stored at -20 C. Avoid repeated freezethaw cycles for maximal stability. How many Western blots can one do with 100 ug total protein lysate? Answer: You can expect to use 100 ug for approximately experiments. This depends on the specific protein expression level in your lysate, and the sensitivity of the antibody used for detection. We recommend starting your experiments with 5 ug total protein per lane of the acrylamide gel. If the SDS-reconstituted lysates have been aliquotted and frozen, do I have to boil them before loading onto an acrylamide gel? Answer: Yes, it is recommended. This will assure that the protein is completely denatured. However, boiling transmembrane proteins in SDS sample buffer tends to induce protein aggregation. We suggest that you incubate the overexpression lysates of these proteins with 1X SDS sample buffer at room temperature for 30 minutes prior to loading. What source do you use for the predicted molecular weight of the VERIFY antigens? Answer: We mainly use the SWISS-PROT website to predict the molecular weight of the unprocessed protein. Why do I sometimes see more than one reactive band with a VERIFY antigen standard in Western blot experiments? Answer: This happens from time to time because of posttranslation modifications, protein degradation, etc., and is not necessarily an indication that there is a problem with your lysates or antibody. Does OriGene sell protein-specific antibodies that can be used with these standards? Answer: OriGene does sell some protein-specific antibodies which have been validated in Western blot applications. Please check our website at for a complete list of antibodies available. What should I do if I can t find the specific VERIFY antigen standard I m interested in? Answer: OriGene is routinely adding new lysate products to our collection on a regular basis. Please check our website frequently or fill out the feedback form on our website at to help us prioritize new product releases. OriGene will contact you as soon as the lysate product you requested is available. Does OriGene sell other reagents for Western blot applications? Answer: Yes, in addition to gene-specific antibodies, OriGene also offers an HRP labeled goat anti-mouse secondary antibody (product # TA100015), chemiluminescent reagents (product # TA100016), and a MYC/DDK tagged molecular weight marker (product #MWM1001). See our website at origene.com/antibody/wbreagents.mspx for more details. If Western blot analysis shows that the immunoreactive band is different from your predicted molecular weight, does it mean that I did not get the correct antigen standard for my gene? Answer: The predicted molecular weight is calculated from the primary amino acid composition. There could be some discrepancy from the actual molecular weight, if there are post-translational modifications or known proteolytic activity. In this case, OriGene strongly suggests researchers check relevant literature on that particular protein GeneDex_V3_4_12-8.indd 6 12/9/08 11:55:10 AM

7 Three ORF clone vectors you shouldn t be without! You probably are aware that OriGene s entire TrueClone collection (the largest existing group of full-length human cdna clones) is available as both the native cdna and in the highly useful TrueORF format. But you may not know that the TrueORF line continues to expand and improve. Now clones in a GFP-tagged expression vector are available as standard inventory items (no longer a custom subcloning order), and we re launching an inducible vector (ptune) as well. As always, all TrueORFs are available as standard orders in pcmv6-entry, a vector with C-terminal Myc and DDK* tags that serves as both an independent expression vector and as the entry vector for OriGene s PrecisionShuttle system. Inducible expression The ptune Inducible Gene Expression System is a mammalian overexpression vector that has complete or graduated control of expression of the epitope tagged ORF that it contains. This system couples repressor proteins and an RNAi target design to effectively turn off any gene (Deans et al.; see Figure 1). Unlike other inducible vectors using either the tetracycline (tet) repressor or small interfering RNAs which have a certain degree of leakiness, the ptune Inducible vector combines repression at a lactose operon (lac) with a tet-controlled promoter using RNA interference as a means to achieve tight inhibition. The advantages of this inducible Gene Expression System are: tight regulation of gene expression graduated control of gene expression using different IPTG concentrations reversibility of switch universality can be used with any gene of interest Myc and DDK tagged protein expression compatibility with OriGene s 37,000 (and always increasing) TrueORF cdna clones Continued on Page 8 Figure 1 A. Switch OFF (No Induction) CMV TetR CMV Lac I TetO shrna RSV GFP shrna Target LacO LacO B. Switch ON (Induced) IPTG CMV TetR CMV Lac I TetO shrna RSV GFP shrna Target LacO LacO Figure 1. Schematic of ptune Inducible vector. In this figure, the GFP ORF is used as an example. In ptune clones, your gene of interest will be cloned in the position of the GFP ORF shown here. (A) In the OFF state, repressor proteins (Lac I) are expressed and exert an effect on the lac operator sites downstream of the CMV and RSV promoters. This results in repression of gene expression (TetR and GFP). As the result of repression of TetR, shrna is transcribed by the U6 promoter and works on the target sequence located 3 to the GFP cassette, preventing any potential residual gene expression. (B) In the ON state, isopropyl-b-thiogalactopyranoside (IPTG) binds to the Lac I proteins, resulting in a conformational change in the repressor proteins. The addition of IPTG causes a release of repressor proteins from the lac operator sites, allowing transcription of tetr and the gene of interest. Tet repressor proteins bind to the tet operator site located in the U6 promoter of the RNAi, shutting down the transcription of the inhibitory shrna GeneDex_V3_4_12-8.indd 7 12/9/08 11:55:11 AM

8 Continued from Page 7 Seeing is believing When GFP is fused to an ORF of interest, the expressed protein retains normal activity so that localization, movement and other activities of the protein can be inferred by analysis of the GFP fluorescence (Wang and Hazelrigg, 1994). Now that all TrueORF clones are in stock with GFP tags (no custom subcloning required), you can you see sooner than before. OriGene s flagship TrueORF clones are all available in a green fluorescence protein (GFP)-tagged vector, enabling easy monitoring of transfection and protein localization. Over 25,000 human open reading frame (ORF) clones and 12,000 mouse ORF clones can be ordered in the pcmv6-ac-gfp vector. The GFP marker can be used to localize the expressed protein in the cell (Figure 2) or simply to confirm or quantitate transfection efficiency. Nothing quite like the original The first TrueORF vector released was pcmv6-entry, still one of our most popular vector options. Not only can pcmv6-entry be used immediately upon delivery as a transfectable, tagged mammalian expression vector, it is also the entry vector for OriGene s twenty different destination vectors. This plasmid features C-terminal Myc and DDK* tags, enabling imaging and quantitation of protein expression using anti-tag antibodies (also available from OriGene; product #TA100014). If you re looking for a different tag (His, HA, or some combination of two tags), or would prefer an N-terminally tagged protein, then pcmv6-entry is still the best place to start. A simple cut-andpaste system will shuttle any ORF from pcmv6-entry to a destination vector of your choice. And, oh, do we have choices! See our website at for a complete list of available destination vectors, including a description of tags and selection markers for each plasmid. Figure 2 A. B. C. D. IL-6 IL-6 + Figure 2. TrueORF clones in pcmv6-ac-gfp express a visually tagged protein, useful for protein localization studies. A and B. HEK293 cells were transfected with pcmv6-an-gfp-stat3 (PS100019, RC215836) hours post-transfection, STAT3 was visualized directly using a fluorescent microscope (Olympus FluoView FV1000 confocal microscope) without staining. Upon stimulation with IL-6, STAT3 was activated and then translocated from cytoplasm (panel A) into nucleus (panel B) to regulate cell proliferation. C and D. HEK293 cells were co-transfected with pcmv6-an-gfp-actinb (PS100019, RC203643) and RhoA-Q63L (a constitutively activated RhoA). Actin bundled filopodia and lamellipodia (panel C, green) and actin-stress fibers (panel D, green) were visualized directly as above. The nuclei were stained with Topra-3 (red) GeneDex_V3_4_12-8.indd 8 12/9/08 11:55:12 AM

9 PrecisionShuttle Vectors All of the plasmids in the PrecisionShuttle vector system allow high-level target gene expression in mammalian cells or via in vitro translation in a cell-free system. The plasmids contain the promoter and enhancers of the human cytomegalovirus (CMV) immediate early gene to drive mammalian gene expression, and the T7 promoter for in vitro transcription/translation. An optimal Kozak consensus sequence is included in the plasmid to enhance mammalian cell expression. All TrueORF clones are purified from a single colony and are shipped as a 10 ug quantity (unless otherwise contracted from OriGene). Researchers can immediately use this DNA in their experiments by transfecting the provided purified DNA into target cells. The development of the PrecisionShuttle vector system has gone through a rigorous quality control (QC) process. The entry vector and all destination vectors have been validated for transient and stable mammalian cell transfections using a T-GFP marker (data not shown). What s the big deal about GFP? The 2008 Nobel Prize in Chemistry was awarded pcmv6-entry Tagged PrecisionShuttle Entry Vector Kan r / Neo r pcmv6-ac-gfp Tagged PrecisionShuttle Vector ColE1 SV40 ori HSV TK Poly A Amp Neo r f1 ori f1 ori CMV promoter pcmv6-entry 4929 bp CMV promoter pcmv6-ac-gfp 6590 bp PolyA signal SV40 ori MCS myc tag DDK tag PolyA signal ColE1 ORF ORF GFP ptune Inducible PrecisionShuttle Vector T7 Promoter Sgf I Asc I Rsr II Mlu I Not I Xho I T7 Promoter Sgf I Asc I Rsr II Mlu I to Drs. Osamu Shimomura, Martin Chalfie, and Roger Tsien for the discovery and development of the green fluorescent protein, GFP. Dr. Shimomura was credited for the discovery of GFP and characterization of its fluorescent properties. Dr. Chalfie demonstrated that fluorescent GFP Lac 0 CMV U6 / Tet 0 shrna Lac I Tet r ptune 12.1 kb Neo r shrna target DDK tag myc tag Lac 0 Sac II Fse I Pme I Xho I Asc I Sgf I can be expressed independently in E. coli systems, opening the door for its use as a universal CMV Amp r Ori RSV genetic tag. Dr. Tsien has developed numerous variants of GFP extensively used in research today that fluoresce with increased brightness, photostability and/or different emission spectra than the original GFP protein. References 1. Deans TL, Cantor CR, Collins JJ. A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell Jul 27;130(2): Wang S, Hazelrigg T. Implications for bcd mrna localization from spatial distribution of exu protein in Drosophila oogenesis. Nature Jun 2;369(6479): *Peptide sequence of the DDK-tag (Flag ): N-DYKDDDDK-C Flag is a registered trademark of Sigma-Aldrich GeneDex_V3_4_12-8.indd 9 12/9/08 11:55:13 AM

10 Protocols for ORF vectors Transfer of ORF from pentry to any destination vector To transfer the protein-coding region from the TrueORF Entry Vector (donor) to a PrecisionShuttle destination vector (recipient), choose the appropriate destination vector with the desired tag options (see for a complete list of vectors). The transfer protocol between TrueORF vectors is shown schematically and detailed below*. Neo r /Kan r Digestion with Sgf I and Mlu I Neo r /Kan r Sgf I TrueORF Entry-Vector Sgf I TrueORF Entry-Vector Amp r Protein ORF Neo r Mlu I Myc-DDK Tag Protein ORF Mlu I Tag Sgf I Precision Shuttle Destination- Vector Amp r Amp r Neo r Neo r Ligation Precision Shuttle Destination- Vector Tag Sgf I Digestion with Mlu I and CIP Tag Sgf I Precision Mlu I Shuttle Destination- Vector Transformation and Selection with Ampicillin Protein ORF Mlu I Mlu I 1. Digest the TrueORF entry clone: Volume 10X restriction buffer** µl Sgf I (10 U/µl) 0.6 µl Mlu I (10 U/µl) 0.6 µl nuclease-free water 13.8 µl TrueORF entry vector ( ng) 3 µl Total volume 0 µl Incubate at 37 C for 1 hour. 2. Digest the TrueORF destination vector: Volume 10X restriction buffer** µl Sgf I (10 U/µl) 0.6 µl Mlu I (10 U/µl) 0.6 µl nuclease-free water 14.8 µl TrueORF destination vector (200ng) µl Total volume 0 µl Incubate at 37 C for 1 hour. Add 0.4 µl calf intestine phosphatase to the digestion, and continue to incubate at 37 C for an additional 30 minutes. * For the 4% of the clones that have internal Sgf I or Mlu I sites, please use the appropriate combination of restriction sites as recommended by OriGene ** NEB buffer 3 has been shown to work well with dual digestion of Sgf I and Mlu I. 3. Purify the digestions using commercial PCR purification columns and elute each in 20 ul 10 mm Tris. 4. Set up a ligation reaction: Volume 10x T4 DNA ligation buffer 1 µl T4 DNA Ligase (4U/µl) 0.75 µl nuclease-free water 3.25 µl digested DNA from Step 1 µl digested DNA from Step 2 3 µl Total volume 10 µl Incubate the ligation reaction at room temperature for 1 hour. 5. Transform the ligation reaction into high-efficiency, competent E. coli cells ( 1x10 8 CFU/µg DNA) following the appropriate transformation protocol. Plate the transformants on LB-agar plates supplemented with 100 µg/ml ampicillin GeneDex_V3_4_12-8.indd 10 12/9/08 11:55:14 AM

11 6. Pick at least four colonies for subsequent DNA purification and screening. Amplify and purify the selected clone(s) by growing overnight in liquid LB-amp media, then isolating the DNA using standard plasmid purification procedures. 7. Confirm the insert by restriction digestion and/or vector primer sequencing using the provided VP1.5 for 5 end sequencing and XL39 for 3 end sequencing. Cloning into p-tune inducible ORF vector (when no Sgf I or Xho I sites exist in the ORF) OriGene provides a TrueORF custom subcloning service for our all of our customers. Or you can choose to shuttle an ORF from the OriGene entry vector into the ptune Inducible vector yourself. If you choose to do the shuttling yourself, we recommend the following protocol. Please note that this is only recommended if Sgf I and Xho I site are absent from your target ORF. Otherwise, we recommend purchasing the TrueORF in pcmv6-entry and shuttling the insert into ptune vector. (See subsequent protocol for details.) 1. Primer design and PCR amplification The open reading frame (ORF) of the clone must be PCR amplified in order to append cloning sites to the 5 and 3 ends of the sequence. Add the target sequences of the selected restriction enzymes to the forward and reverse PCR primers; examples are shown below. Forward primer with Sgf I 5 GAGGCGATCGCCNNNNNNNNNNNNNNNNNNNNNNN 3 (Ns represent the sequence of the ORF beginning with the start codon, ATG) Reverse primer with Xho I 5 GCGCTCGAGNNNNNNNNNNNNNNNNNNNNNNNN 3 (Ns represent the last 8 codons of the ORF in reverse complement orientation. These do not include the stop codon that must be removed to generate a fusion protein for C-terminally tagged vectors. We recommend using a full-length cdna clone as the template for ORF cloning. The success rate is rather low when a cdna pool is used as the template for a PCR cloning reaction. When the GC content of an ORF (or a region of the ORF longer than 100 bp) is above 75%, a special PCR buffer with DMSO or other additive should be used to increase the success rate. PCR reaction setup: Volume 5X PCR buffer 4.0 µl dntps (2.5 mm each) 1.6 µl Phusion polymerase (2U/µl) 0.2 µl nuclease-free water 11.0 µl forward primer (10 µm) 0.6 µl reverse primer (10 µm) 0.6 µl cdna template ( ng plasmid).0 µl total volume 0.0 µl All the components should be kept on ice. When setting up multiple reactions, a master mix can be prepared without cdna template or primers. After aliquotting the master mix, the cdna template and primers can be added individually to each tube. PCR cycling conditions: Temperature/Time Cycles 95 C/1 min 1 95 C/10 sec, 62 C/20 sec, 72 C/4 min 95 C/10 sec, 60 C/20 sec, 72 C/4 min 95 C/10 sec, 58 C/20 sec, 72 C/4 min 95 C/10 sec, 56 C/20 sec, 72 C/4 min C/10 min 1 4 C Hold The optimum temperature for annealing should be C. The extension time depends upon the length of the ORF. The conditions above are generally used for ORFs from 500 bp bp. 2. Digesting the donor product and the recipient vector Confirm that the size of the amplification product is correct by agarose gel electrophoresis, and purify the remainder of the reaction using a purification column or similar method. Elute the DNA from the purification column in 26 µl of 10 mm Tris buffer. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate for your cloning strategy. Continued on Page 12 The recommended PCR polymerase and buffer are available from New England Biolabs (Phusion High-Fidelity PCR Kit, F-553S) GeneDex_V3_4_12-8.indd 11 12/9/08 11:55:14 AM

12 Continued from Page 11 Incubate the ligation reaction at 12 C overnight. Volume (µl) 10X restriction buffer 3.0 Sgf I (10U/µl) 0.6 Xho I (10U/µl) 0.6 purified PCR product 6.0 total volume ~ 30.0 Mix well, and incubate at 37 C for 1 hour. Purify the digestion reaction using a purification column and elute in 18 µl of 10 mm Tris buffer. Digest ptune Inducible Vector with the restriction enzymes corresponding to the sequences added to the ORF. Volume (µl) 10X restriction buffer 3.0 Sgf I (10U/µl) 0.8 Xho I (10U/µl) 0.8 ptune Inducible Vector 10.0 nuclease-free water 15.4 total volume 30.0 Incubate at 37 C for 1.5 hr, then add 0.5 µl alkaline phosphatase, and continue the incubation at 37ºC for another 30 min. A two hour digestion is recommended to ensure that the vector is completely digested. Dephosphorylation of the digested vector is essential to eliminate self-ligation. Purify the desired vector fragment by running the digestion reaction on an agarose gel, and isolating the appropriate band using a gel purification column. Elute the digested plasmid vector in 40 µl of 10 mm Tris buffer. 3. Ligation Set up a ligation reaction with the purified insert and vector fragments: Volume (µl) 10X T4 DNA ligase buffer 1.0 nuclease-free water 3.5 T4 DNA ligase (4U/µl) 0.5 purified, digested ptune Inducible Vector fragment.0 purified, digested PCR product 3.0 total volume Transformation Transform 1 µl of the ligation mixture using 20 µl high efficiency competent E. coli cells (ideally at 1x10 8 CFU/ug). Following transformation, resuspend the cells in 200 ul LB. Plate the entire transformation reaction on a standard LBagar plate containing 100 µg/ml ampicillin. Incubate at 37 C overnight. Pick at least 4-8 independent colonies from each ligation. Confirm the insert by restriction digestion and/or vector primer sequencing using the provided ptune-f for 5 end sequencing and ptune-r for 3 end sequencing. Cloning into the ptune inducible ORF vector (when Sgf I or Xho I sites exist in the ORF) If even a single Xho I site exists in the ORF, then this ORF cannot be directly transferred into ptune. The ORF must first be cloned into pcmv6-entry using Sgf I and Mlu I, then shuttled from pcmv6-entry into ptune. Depending on what sites are present in the ORF, the shuttling from pcmv6-entry into ptune can be accomplished using Sgf I at 5 end and Pme I or Fse I or Sac II at 3 end. (This method transfers the entire ORF and the Myc and Flag tags from pcmv6-entry into ptune.) Part I. Cloning of ORF into pcmv6-entry 1. Primer design and PCR amplification The open reading frame (ORF) of the clone must be PCR amplified in order to append cloning sites to the 5 and 3 ends of the sequence. Add the target sequences of the selected restriction enzymes to the forward and reverse PCR primers; examples are shown below. Forward primer with Sgf I 5 GAGGCGATCGCCNNNNNNNNNNNNNNNNNNNNNNN 3 (Ns represent the sequence of the ORF beginning with the start codon, ATG) Reverse primer with Mlu I 5 GCGACGCGTNNNNNNNNNNNNNNNNNNNNNNNN 3 (Ns represent the reverse complement of the ORF sequence starting with the second-to-last codon; the stop codon must be removed to generate a fusion protein for C-terminally tagged vectors, GeneDex_V3_4_12-8.indd 12 12/9/08 11:55:14 AM

13 If the recognition sites for Sgf I or Mlu I are present internally in the ORF, another rare cutter such as Asc I, Rsr II,Not I or Xho I can be used in the cloning strategy. In these cases, the sequences of these alternate restriction sites should be used in place of Sgf I and/or Mlu I (examples below). This same primer design strategy described above should be used for design of other primers. The Ns in the forward primer represent the sequence of the ORF beginning with the start codon, ATG. The Ns in the reverse primers represent the reverse complement of the ORF sequence starting with the second-to-last codon for C-terminally tagged vectors. Forward primer with Asc I: 5 GCCGGCGCGCCNNNNNNNNNNNNNNNNNNNNNNN 3 Reverse primer with Rsr II: 5 GCGTCGGACCGCTNNNNNNNNNNNNNNNNNNNNNNNN 3 Reverse primer with Not I: 5 GCGACGCGGCCGCTCACGCGTNNNNNNNNNNNNNNNNNNNNNN NN 3 Reverse primer with Xho I 5 GCGCTCGAGNNNNNNNNNNNNNNNNNNNNNNNN 3 We recommend using a full-length cdna clone as the template for ORF cloning. The success rate is rather low when a cdna pool is used as the template for a PCR cloning reaction. When the GC content of an ORF (or a region of the ORF longer than 100 bp) is above 75%, a special PCR buffer with DMSO or other additive should be used to increase the success rate. The recommended PCR polymerase and buffer are available from New England Biolabs (Phusion High-Fidelity PCR Kit, F-553S). PCR reaction setup: Volume 5X PCR buffer 4.0 µl dntps (2.5 mm each) 1.6 µl Phusion polymerase (2U/µl) 0.2 µl nuclease-free water 11.0 µl forward primer (10 µm) 0.6 µl reverse primer (10 µm) 0.6 µl cdna template ( ng Plasmid).0 µl total volume 0.0 µl All the components should be kept on ice. When setting up multiple reactions, a master mix can be prepared without cdna template or primers. After aliquotting the master mix, the cdna template and primers can be added individually to each tube. PCR cycling conditions: Temperature/Time Cycles 95 C/1 min 1 95 C/10 sec, 62 C/20 sec, 72 C/4 min 95 C/10 sec, 60 C/20 sec, 72 C/4 min 95 C/10 sec, 58 C/20 sec, 72 C/4 min 95 C/10 sec, 56 C/20 sec, 72 C/4 min C/10 min 1 4 C Hold The optimum temperature for annealing should be C. The extension time depends upon the length of the ORF. The conditions above are generally used for ORFs from 500 bp bp. 2. Digesting the donor product and the recipient vector Confirm that the size of the amplification product is correct by agarose gel electrophoresis, and purify the remainder of the reaction using a purification column or similar method. Elute the DNA from the purification column in 26 µl of 10 mm Tris buffer. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate for your cloning strategy. Volume (µl) 10X restriction buffer 3.0 Sgf I (10U/µl) 0.6 Mlu I (10U/µl) 0.6 purified PCR product 6.0 total volume ~ 30.0 Mix well, and incubate at 37 C for 1 hour. Purify the digestion reaction using a purification column and elute in 18 µl of 10 mm Tris buffer. Digest pentry with the restriction enzymes corresponding to the sequences added to the ORF. Continued on Page GeneDex_V3_4_12-8.indd 13 12/9/08 11:55:14 AM

14 Continued from Page 13 Volume (µl) 10X restriction buffer 3.0 Sgf I (10U/µl) 0.8 Mlu I (10U/µl) 0.8 pcmv6-entry 10.0 nuclease-free water 15.4 total volume 30.0 Part II. Transfer of ORF from pcmv6-entry to ptune inducible vector The ORF of a C-terminally tagged protein is followed by a double tag (Myc and DDK) including a short spacer (5-6 amino acid residues). The MCS of pcmv6-entry and ptune vectors are similar, except that some cloning sites in pcmv6-entry (such as Mlu I, Rsr II and Not I) are not unique restriction sites in ptune vector. The ORF transfer protocol from pcmv6-entry vector to ptune vector is detailed below. Incubate at 37 C for 1.5 hr, then add 0.5 µl alkaline phosphatase, and continue the incubation at 37ºC for another 30 min. A two hour digestion is recommended to ensure that the vector is completely digested. Dephosphorylation of the digested vector is essential to eliminate self-ligation. Purify the desired vector fragment by running the digestion reaction on an agarose gel, and isolating the appropriate band using a gel purification column. Elute the digested plasmid vector in 40 µl of 10 mm Tris buffer. 3. Ligation Set up a ligation reaction with the purified insert and vector fragments: Volume (µl) 10X T4 DNA ligase buffer 1.0 nuclease-free water 3.5 T4 DNA ligase (4U/µl) 0.5 purified, digested pcmv6-entry vector fragment 2.0 purified, digested PCR product 3.0 total volume 10.0 Incubate the ligation reaction at 12 C overnight. 4. Transformation Transform 1 µl of the ligation mixture using 20 µl high efficiency competent E. coli cells (ideally at 1x10 8 CFU/ug). Following transformation, resuspend the cells in 200 ul LB. Plate the entire transformation reaction on a standard LBagar plate containing 100 µg/ml kanamycin. Incubate at 37 C overnight. Pick at least 4-8 independent colonies from each ligation. Confirm the insert by restriction digestion and/or vector primer sequencing. 1. Digesting the donor and the recipient vectors Digest pcmv6-entry containing your ORF: Volume (µl) 10X restriction buffer.0 Sgf I (10U/µl) 0.6 Sac II or Fse I or Pme I (10U/µl) 0.6 ORF cdna clone in pcmv6-entry ( ng) 3.0 nuclease-free water 13.8 total volume 0.0 Incubate at 37 C for 1 hour. Digest ptune inducible vector: Volume (µl) 10X restriction buffer.0 Sgf I (10U/µl) 0.6 Sac II or Fse I or Pme I (10U/µl) 0.6 ptune inducible vector (200ng) 3.0 nuclease-free water 13.8 total volume 0.0 Incubate at 37 C for 1 hour. Add 0.4 µl calf intestine phosphatase to the digestion, and continue to incubate at 37 C for an additional 30 minutes. Purify the digestion using a commercial PCR purification column and elute in 20 ul 10 mm Tris. 2. Ligation Set up a ligation reaction: Volume (µl) 10X T4 DNA ligase buffer 1.0 nuclease-free water 3.25 T4 DNA ligase (4U/µl) 0.75 digested ORF insert in pcmv6-entry vector 2.0 digested ptune vector fragment 3.0 total volume 10.0 Incubate the ligation reaction at 12 C overnight GeneDex_V3_4_12-8.indd 14 12/9/08 11:55:15 AM

15 3. Transformation Transform the ligation reaction into high-efficiency, competent E. coli cells (>1x10 8 CFU/µg DNA) following the appropriate transformation protocol. Plate the transformants on LB-agar plates supplemented with 100 µg/ml ampicillin. Pick at least six colonies for subsequent DNA purification and screening. Confirm the insert by restriction digestion and/or vector primer sequencing using the provided ptune-f for 5 end sequencing and ptune-r for 3 end sequencing. 2. Preparation of the MegaTran 1.0/DNA complexes Prepare immediately prior to transfection. For each well to be transfected, dilute 1 µg of DNA into 100 ul of Opti-MEM I (Gibco 51985). Vortex gently. Add 3 ul of MegaTran 1.0 for every µg of DNA into the DNA/Opti-MEM solution (not the reverse order) and vortex the solution immediately for 10 seconds. Incubate for 10 minutes at room temperature. Note: We recommend starting with the ratios of MegaTran 1.0 and DNA listed in the Table; however, subsequent optimization may further increase the transfection efficiency. 3. Transfection Protocol for transient transfection (for any TrueORF vector) A sample protocol is listed here for experiments performed in 24-well plates. If performing experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. See Table for more details. 1. Preparation of cells Plate ~5x10 4 adherent cells or ~5x10 5 suspension cells per well 24 hours prior to transfection. Gently add the MegaTran 1.0/DNA mixture from step 2 to each well (already containing about 900 ul culture medium). Generally, the volume of the MegaTran 1.0/ DNA mixture should represent 1/10 of the total volume of the culture medium. Gently rock the plate to achieve even distribution of the complexes. Incubate at 37 C for hours. Note: The above incubation is designed for transfection without a media change. If a media change is preferred, incubate the transformation mixture on the cells for 30 minutes (if centrifugation is possible) or for 3-4 hours (if centrifugation is not possible). Replace the media with the fresh complete growth media. Incubate for hours. Continued on Page 16 Table. Recommended starting transfection conditions for MegaTran 1.0 culture vessel growth area cell number DNA (μg) MegaTran 1.0 (μl) cm 2 /well (adherent cells) 96-well plate x well plate x well plate x well plate x well plate x mm plate x mm plate x GeneDex_V3_4_12-8.indd 15 12/9/08 11:55:15 AM

16 Continued from Page 15 Protocol for stable transfection (for any TrueORF vector) Perform a transfection as described above (Protocol for transient transfection). Twenty-four hours post-transfection, passage the cells (at 1:10 or higher dilution) into fresh growth medium containing a selective medium such as G418, a neomycin analog. A mock transfection should be performed in parallel as a control. Grow and passage the cells as necessary, maintaining selection pressure by keeping the selective agent in the growth medium. After 1-2 weeks, a large number of the cells will be killed; the cells that remain growing in the selective medium have retained the expression plasmid, which stably integrates into the genome of the targeted cells. Protocol for inducing expression from ptune 1. Prepare IPTG Prepare 1M stock of IPTG and dilute it with sterilized water. 2. IPTG Induction For transient transfections, add the desired amount (2.5 um -1 mm) of IPTG to the cells 1-2 hours after the transfection was performed. For stable transfections, change the medium daily, adding IPTG at the desired dilution to the fresh medium. But have you seen us lately? OriGene Technologies is proud to unveil our newly redesigned website, which offers a comprehensive overview of the diverse lines of genetic research tools and reagents that we offer. This website clearly demonstrates OriGene s attitude as Your Gene Company. As genomics scientists ourselves, we recognize that our customers are employing an ever more sophisticated strategy in their research agenda. A cdna clone customer is also involved in RNAi experiments, qpcr transcript profiling, and antibody validation. OriGene s products are organized into product lines, with new pages reflecting the expansion of our antibody holdings and the addition of new products, such as our protein and cell lysates and qpcr primers. This allows a customer to zero in on the specific product they re looking for, while maintaining the ability to search for all products relevant to any gene by using that gene s accession number, keyword, or sequence. Moreover, the newly launched website is easy to navigate, without an endless series of redirection pages. As a young life science tools company, OriGene always strives to present most comprehensive product information on our website, says Walter Tian, OriGene s vice president of marketing. The new website allows us to better illustrate OriGene s gene centric philosophy. With significant improvements to our online search engine, we are able to significantly reduce search failure rates and provide appropriate product solutions. I am extremely happy with these upgrades. This is only the beginning. Researchers can expect more and better features from our website. Of course, some things haven t changed. The website continues to be a useful source of information: protocols for common genomics techniques, frequently asked questions about our products, quality control and product specifications, downloadable manuals and technical articles. We re constantly updating our list of citations that provide peerreviewed, independent confirmation of the quality of our products. And we re still asking for your input: give us your suggestions on how we can better serve your research genomics needs, whether by changing a feature of our website or creating a new product tailored to your specific project. Contact us anytime through our new website at or call us at ( outside the US). We re here to help you GeneDex_V3_4_12-8.indd 16 12/9/08 11:55:15 AM

17 Frequently Asked Questions about pcmv6-entry, pcmv- AC-GFP, and ptune Why should I use OriGene s TrueORF clones? Answer: All TrueORF Clones are derived from OriGene s unique TrueClone Collection, and were isolated from high quality cdna libraries made from a variety of tissues. TrueORF Clones allow customers to directly apply these expressionready, tagged ORF clones to experiments designed for protein expression, purification, protein-protein interaction and stable clone selection. pcmv6-entry clones also serve as the entry clone for the ptune Inducible system and allow easy construction of an inducible, tagged ORF. pcmv-ac-gfp allows for easy visualization of successful transfection, and is ideal for studies where subcellular localization or quantitation of transfection is important. All TrueORF and ptune Inducible vectors share similar multiple cloning sites (MCS). Customers can easily shuttle the cdna of a TrueORF clone to the ptune Inducible vector. What are the functional aspects of the pcmv6-ac-gfp vector? Answer: Like all OriGene vectors, the CMV promoter drives heterologous expression of the specified open reading frame (ORF), which is in-frame with green fluorescent protein (GFP) at the C-terminus. Such fluorescence permits the positive identification of mammalian cells successfully transfected with the plasmid. The neomycin resistance gene is also expressed downstream of the SV40 promoter in the same vector and permits positive selection of transfected cells, as well as stable cell line production. For bacterial amplification, the ampicillin resistance gene is engineered on the opposite strand. OriGene s GFP is listed as TurboGFP. How is this different from other available GFPs? Answer: TurboGFP is a fully licensed, 26 kda protein product from Evrogen JSC that works well in standardized GFP assays. The peak excitation wavelength is 482 nm and the peak emission wavelength is 502nm. TurboGFP yields 112% of the brightness compared to egfp, and has no known cell toxicity. It is an isoform of the naturally occurring protein from Pnotillina plumata that is optimized for rapid labeling of cells/organelles and tracking of promoter activity. It is a perfect choice for monitoring transient protein expression. How many amino acids are present in the linker between my protein and GFP? Answer: To accommodate the Mlu I cloning site, which maintains the proper reading frame, this vector uses threonine and arginine. This is far fewer amino acids than in other recombination based shuttling systems. What is the advantage of the ptune vector? Answer: The ptune inducible system is an engineered, tunable genetic switch that tightly regulates gene expression in mammalian cells, allowing you to temporally control expression of the ORF after transfection into mammalian cells. The ptune vector combines repression at a lactose operon (lac) with a tet-controlled promoter using RNA interference as a means to achieve tight inhibition. What restriction enzymes should I use to shuttle ORFs if Sgf I, Mlu I, or Xho I sites are present in my ORF? Answer: While 96% of all human and mouse ORFs can use the Sgf I - Mlu I combination for shuttling from pcmv6-entry to a destination vector, some ORFs do contain internal Mlu I site(s). Most of these ORFs with an internal Mlu I site can be transferred using other rare cutters (Rsr II or Not I); recommended digestion strategies are listed for each product. Many ORFs can be cloned directly into ptune. However, if Sgf I or Xho I sites are present in the ORF, the sequence must first be cloned into pcmv6-entry, then shuttled into ptune using Sac II or Pme I or Fse I. Can I transfer large ORFs into ptune? Answer: It has been reported that ORFs larger than 4 Kb are unstable in other, recombination-based systems. Several ORFs ranging from 0.7 to 10 kb have been successfully cloned into this inducible vector. ORFs larger than 10 kb have not been tested. Can I purchase empty vectors without any open reading frame? Answer: Yes, lyophilized 10ug aliquots are available using the catalog numbers PS for pcmv6-entry, PS for pcmv6-ac-gfp, and PS for ptune. Continued on Page GeneDex_V3_4_12-8.indd 17 12/9/08 11:55:15 AM

18 Continued from Page 17 What does it mean that these vectors are compatible with OriGene s other Precision Shuttle vectors? Answer: The extremely rare cutting enzymes, Sgf I and Mlu I, were used in the cloning of over 99% of OriGene s TrueORFs, and these same sites are available in over 20 different destination vectors allowing for easy shuttling of ORFs to and from each without the requirement for full sequence confirmation. Sequencing of the cloning sites is always encouraged for those rare cases when a cloning artifact occurs (usually a deletion due to base hydrolysis prior to ligation). Has OriGene fully sequenced all TrueORF clones? Answer: Not always. When transferring the cdna into the TrueORF Entry Vector, OriGene always uses fully sequenced plasmids as templates and Phusion High-Fidelity DNA Polymerase (New England Biolabs), which has a mutation rate less than 4 x This ensures the highest fidelity of every TrueORF clone. After cloning into the entry vector, each of OriGene s TrueORF clones was sequenced at both the 5 and 3 ends, and the resulting sequence was matched to the corresponding reference sequence. For many ORFs 1 Kb or less in length, the 5 and 3 sequencing reads have covered the full ORF. For longer cdnas, the ORF was not fully covered by sequencing reads. What sites should I use to transfer a TrueORF clone into the Gateway system? Answer: There are multiple sites in pcmv6-entry than can be used to move the insert of a TrueORF clone into any of Gateway s entry vectors (pentr-1a, -2B, -3C, -4, and -11). These sites are EcoR I, Sal I, BamH I and Kpn I at the 5 end, and Not I at the 3 end. Do TrueORF clones exactly match the reference gene sequence? Answer: All TrueORF clones are guaranteed to match the ORF of the corresponding gene sequences as published on OriGene s website. However, some clones may contain nucleotide changes compared to the published reference sequences. This is due to SNPs (single nucleotide polymorphisms) reflecting the unique differences from genes expressed in different tissues and different individuals. Published references may represent a different SNP than the OriGene transcript. Should a specific SNP be required, this can be contracted from OriGene at an additional charge. Good data and free merchandise it s a win/win situation! Have you published a journal article describing research utilizing an OriGene product? First, congratulations on your publication! Second, we d like to recognize your accomplishment with a small token of our appreciation of you as a valued OriGene customer. For every citation you ve authored that involves an OriGene clone, array, shrna construct, or other reagents from OriGene, we would like to offer you the choice of a product discount or an OriGene green genes T-shirt. Send your name, complete mailing address, and T-shirt size (choose M, L, or XL) and a PDF of your article to cdna@ origene.com with the subject line citation request. Indicate your preference for either a product discount or T-shirt. Offer good while supplies last. Shirts and sizes are subject to availability. What does your disclaimer mean? Answer: OriGene s disclaimer for the TrueORF clones reads as follows: Our molecular clone sequence data has been matched to the accession number below as a point of reference. Note that the complete sequence of our molecular clones may differ from the sequence published for this corresponding accession number, e.g., by representing an alternative RNA splicing form or single nucleotide polymorphism (SNP). The NCBI RefSeq mrna sequences are continuously being revised, as some may have been derived from aberrantly spliced transcripts or generated by incorrect prediction of intron-exon junctions in silico. These sequences are therefore used only as a reference and not as a standard. OriGene s clones are isolated from full-length cdna libraries and may differ from the reference sequence for this reason. What is the TrueORF Guarantee? Answer: OriGene warrants that the product will meet specifications listed. At OriGene s discretion, free replacement of any non-conforming product will be made if OriGene is notified within 30 days of product receipt. If you experience any difficulty with any OriGene product, please contact our Technical Support Staff at , or outside the US GeneDex_V3_4_12-8.indd 18 12/9/08 11:55:16 AM