Lenti-X
===LentiCRISPRv2构建说明书
LentiCRISPRv2 and lentiGuide-Puro: lentiviral CRISPR/Cas9 and single guide RNA CRISPR (C lustered R egularly I nterspaced S hort P alindromic R epeats) is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Lentiviral CRISPR/Cas can infect a broad variety of mammalian cells by co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA (sgRNA) to facilitate genome editing (Shalem*, Sanjana*, et al., Science 2014). Protocols for cloning into the lentiviral transfer plasmid and general considerations for producing lentivirus are described below. Separate protocols are available for amplifying the genome-scale CRISPR knock-out (GeCKO) libraries. This protocol is for creating individual lentiviral CRISPR plasmids targeting a single genomic locus.lentiCRISPRv2 (one vector system): This plasmid contains two expression cassettes, hSpCas9 and the chimeric guide RNA. The vector can be digested using BsmB I, and a pair of annealed oligos can be cloned into the single guide RNA scaffold. The oligos are designed based on the target site sequence (20bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence, as shown on the next page.lentiGuide-Puro (two vector system): This plasmid expressed only the chimeric guide RNA. It does not contain Cas9. Please use lentiCas9-Blast (a separate lentiviral construct that delivers hSpCas9 and blasticidin resistance) to first integrate Cas9 into your cell line. The lentiGuide-Puro vector can be digested using BsmB I, and a pair of annealed oligos can be cloned into the single guide RNA scaffold. The oligos are designed based on the target site sequence (20bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence, as shown on the next page.Which vector to use: lentiCRISPRv2 is identical to the original lentiCRISPRv1 but produces nearly 10X higher titer virus. lentiGuide-Puro produces >100X higher titer virus over lentiCRISPRv1 and should be used in cell lines where Cas9 has already been integrated in (e.g. using the separate lentiCas9-Blast lentivirus). For applications where Cas9 cannot first be introduced (e.g. primary cells), lentiCRISPRv2 is recommended. After transduction, use puromycin to select for cells with lentiCRISPRv2 or lentiGuide-Puro.Lentiviral production: Before starting any lentiviral work, please ensure compliance with your Environmental Health and Safety office and government/organization/university. Briefly, to make lentivirus, a transfer plasmid (e.g. lentiCRISPRv2 or lentiGuide-Puro) must be co-transfected into HEK293(F)T cells with the packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260). As a positive control for viral production, we often use a CMV-EGFP lentiviral transfer plasmid (eg. AddGene 19319).Target design notes and online resources: For application of Cas9 for site-specific genome editing in eukaryotic cells and organisms, we have computationally identified suitable target sites for the S. pyogenes Cas9 and calculated most likely off-targets within the genome. Please visit to access these Cas9 target design tools. Complete plasmid sequences, protocols, a discussion forum and additional information can be found at the Zhang Lab GeCKO website: /gecko/ . Citation: Please reference the following publications for the use of this material.Improved lentiviral vectors and genome-wide libraries for CRISPR screening. Sanjana NE*, Shalem O*, Zhang F. Nature Methods (2014).Genome-scale CRISPR-Cas9 knockout screening in human cells. Shalem O*, Sanjana NE*, Hartenian E, Shi X, Scott DA, Mikkelsen T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014). Science, 343, 83-7. DOI: 10.1126/science.1247005Target Guide Sequence Cloning ProtocolIn order to clone the target sequence into the lentiCRISPRv2 or lentiGuide-Puro backbone, synthesize two oligos of the following form. All plasmids have the same overhangs after BsmBI digestion and the same oligos can be used for cloning into lentiCRISPRv2, lentiGuide-Puro or lentiCRISPRv1.Example oligo design : Note that the NGG PAM is not included in the designed oligos. Oligonucleotide ordering tips : Standard de-salted oligos (usually the most inexpensive synthesis) are sufficient forcloning. If not already resuspended, dilute each oligo to 100 µM in sterile water or TE.* * * * *Lentiviral vector digestion, oligo annealing and cloning into digested vector:1. Digest and dephosphorylate 5ug of the lentiviral CRISPR plasmid with BsmB I for 30 min at 37C: 5 ug lentiCRISPRv2 or lentiGuide-Puro 3 ul FastDigest BsmB I (Fermentas) 3 ul FastAP (Fermentas) 6 ul 10X FastDigest Buffer 0.6 ul 100 mM DTT (freshly prepared) X ul ddH 2O 60 ul total2. Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. If BsmBI digested, a ~2kb filler piece should be present on the gel. Only gel purify the larger band . Leave the 2kb band.3. Phosphorylate and anneal each pair of oligos: 1 ul Oligo 1 (100 µM) 1 ul Oligo 2 (100 µM) 1 ul 10X T4 Ligation Buffer (NEB) 6.5 ul ddH 2O 0.5 ul T4 PNK (NEB M0201S) 10 ul total Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).Put the phosphorylation/annealing reaction in a thermocycler using the following parameters: 37o C 30 min 95o C 5 min and then ramp down to 25o C at 5o C/min4. Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.5. Set up ligation reaction and incubate at room temperature for 10 min:X ul BsmB I digested plasmidfrom Step 2 (50ng)1 ul diluted oligo duplex from Step 4 5 ul 2X Quick Ligase Buffer (NEB) X ul ddH 2O 10 ul subtotal1 ul Quick Ligase (NEB M2200S) 11 ul total Also perform a negative control ligation (vector-only with water in place of oligos) and transformation.6.Transformation into Stbl3 bacteria . Lentiviral transfer plasmids contain Long-Terminal Repeats (LTRs) and must be transformed into recombination-deficient bacteria. We use homemade Stbl3 (propagated from Invitrogen C7373-03) and get excellent plasmid yields. Although other RecA- strains may work, we have found the most consistent transformations and yields using Stbl3.。
乐泰X p24快速定量试剂盒使用手册说明书
Takara Bio USA, Inc.Lenti-X™ p24 RapidTiter Kit User ManualCat. No. 632200(111616)Takara Bio USA, Inc.1290 Terra Bella Avenue, Mountain View, CA 94043, USAU.S. Technical Support: ********************United States/Canada Asia Pacific Europe Japan Page 1 of 10Table of ContentsI. Introduction (3)II. List of Components (4)A. Lenti-X p24 Rapid Titer Kit (Cat. No. 632200) (4)III. Storage and Stability (4)IV. Safety Guidelines for Working with Lentiviruses (4)A. Advisory (4)B. Biosafety Level 2 (5)V. Additional Materials Required (5)VI. Assay Techniques & Microtiter Plate Washing (6)A. Automatic Plate Washing (6)B. Manual Plate Washing (6)VII. Protocols: Lenti-X p24 Assay Procedures (6)A. Protocol: Specimen Collection and Storage (7)B. Protocol: Wash Buffer Preparation (7)C. Protocol: Preparing Dilutions for the p24 Standard Curve (0.5 hr) (7)D. Protocol: Assaying Your Lentiviral Supernatants (~4 hr) (7)VIII. Interpretation of Results (9)A. Assay Validation (9)B. Determining Your Virus Titer (9)Appendix A: Troubleshooting Guide (9)Table of FiguresFigure 1. Lentiviral p24 is a virus core/capsid protein (3)Figure 2. The Lenti-X p24 Rapid Titer Kit (3)Figure 3. Lenti-X p24 Rapid Titer Kit standard curve (8)Table of TablesTable 1. Sample data for standard curve (8)I. IntroductionPrinciple of the Lenti-X p24 Rapid Titer AssayThe Lenti-X p24 Rapid Titer Kit allows you to quickly determine the titer of any HIV-1-based lentiviralsupernatant using standard ELISA methods. The wells of the included microtiter plate (12 x 8-well strips) arecoated with an anti-HIV-1 p24 capture antibody, which quantitatively binds the HIV-1 p24 in your test samples (p24 is an abundant HIV-1 virus core/capsid protein [Figure 1]). Specifically-bound p24 is detected in a typical “sandwich” ELISA format using a biotinylated anti-p24 secondary antibody, a streptavidin-HRP conjugate, and a color producing substrate (Figure 2). Color intensity is measured spectrophotometrically to indicate the level of p24 in the samples, which can then be precisely quantified against a p24 standard curve. p24 values can then be correlated to virus titer of packaging cell supernatants.Figure 1. Lentiviral p24 is a virus core/capsid protein. The HIV-1 gag gene encodes p24, which is the major lentiviral capsid protein.The lysis of viral particles is necessary to generate soluble p24 which can then be measured by the Lenti-X p24 Rapid Titer Kit.Figure 2. The Lenti-X p24 Rapid Titer Kit. Lentiviral (HIV-1) p24 core protein in packaging cell supernatants is bound to wells of a microtiter plate coated with HIV-1 p24 capture antibody. The presence of bound p24 in the wells is detected using a biotinylated secondary anti-p24 antibody, a streptavidin-horseradish peroxidase conjugate, and a color-producing substrate. Quantitation is performed bycomparing test samples to a p24 standard curve.II. List of ComponentsA. Lenti-X p24 Rapid Titer Kit (Cat. No. 632200)• 1 each Anti-p24 Coated Plate (96 wells)•100 μl p24 Control (10 ng/ml)• 6 ml Lysis Buffer•12 ml Anti-p24 (Biotin Conjugate)•12 ml Streptavidin HRP•120 ml Wash Buffer (20X)•12 ml TMB Substrate•12 ml Stop SolutionOther•Lenti-X p24 Rapid Titer Kit User Manual (PT5002-1)•Lenti-X p24 Rapid Titer Kit Protocol-at-a-Glance (PT5002-2)III. Storage and Stability•All reagents should be stored at 2–8°C. Do not use reagents beyond the expiration date on the label.•Microtiter Strips. Once opened, microtiter strips may be stored at 2–8°C until the expiration date on the label.Strips must be stored under desiccated conditions. Return unused strips to their original foil pouch along with the sachet of desiccant, and securely reseal the pouch by folding over the open end and securing it with adhesive tape.•Wash Buffer.The working concentration (1X) of wash buffer should not be stored for longer than 3 weeks at 2–8°C. We recommend that fresh wash buffer be prepared before each assay. If the diluted buffer becomesvisibly cloudy or develops a precipitate during storage, discard it and prepared fresh buffer.NOTE: The 20X Wash Buffer normally develops a crystalline precipitate during storage at 2–8°C. This willdissolve upon warming at 37°C.IV. Safety Guidelines for Working with LentivirusesA. AdvisoryFor your safety, and the safety of others around you, it is imperative to fully understand the potentialhazards of working with recombinant lentiviruses and the necessary precautions for their use in thelaboratory.The National Institute of Health and Center for Disease Control have designated recombinant lentivirusesas Level 2 organisms. This requires the maintenance of a Biosafety Level 2 facility for work involvingthis virus and others like it. The VSV-G pseudotyped lentiviruses packaged from the HIV-1-based vectorsdescribed here are capable of infecting human cells. The viral supernatants produced by these lentiviralsystems could, depending on your insert, contain potentially hazardous recombinant virus. Similar vectorshave been approved for human gene therapy trials, attesting to their potential ability to express genes invivo.For these reasons, due caution must be exercised in the production and handling of any recombinantlentivirus. The user is strongly advised not to create VSV-G pseudotyped lentiviruses capable ofexpressing known oncogenes.For more information on Biosafety Level 2 agents and practices, download the following reference:Biosafety in Microbiological and Biomedical Laboratories (BMBL), Fifth Edition (February 2007) HHSPub. No. (CDC) 93-8395. U.S. Department of Health and Human Services Centers for Disease Controland Prevention and NIH. Available on the web at/od/ohs/biosfty/bmbl5/bmbl5toc.htmB. Biosafety Level 2The following is a brief description of Biosafety Level 2. It is neither detailed nor complete. Details of thepractices, safety equipment, and facilities that combine to produce a Biosafety Level 2 are available in theabove publication. If possible, observe and learn the practices described below from someone who hasexperience working with lentiviruses.Important Features of Biosafety Level 2:Practices:•Standard microbiological practices•Limited access to work area•Biohazard warning signs posted•Minimize production of aerosols•Decontaminate potentially infectious wastes before disposal•Use precautions with sharps (e.g., syringes, blades)•Biosafety manual defining any needed waste decontamination or medical surveillance policiesSafety equipment:•Biological Safety Cabinet, preferably a Class II BSC/laminar flow hood (with a HEPAmicrofilter) used for all manipulations of agents that cause splashes or aerosols of infectiousmaterials; exhaust air is unrecirculated•PPE: protective laboratory coats, gloves, face protection as needed postedFacilities:•Autoclave for waste decontamination•Chemical disinfectants for spillsV. Additional Materials RequiredThe following materials, or their equivalents, are required to perform the Lenti-X p24 Rapid Titer Kit assay: •Micropipettes for delivering volumes of 5 µl, 10 µl, 25 µl, 100 µl, and 200 µl. A multichannel pipette is preferred for dispensing reagents into microtiter plates.•Distilled or deionized water• A 37°C ± 1°C incubator•Disposable plastic or glass test tubes; 5 ml and 10 ml capacities•Laboratory glassware: 15 ml and 100 ml beakers; 1 L graduated cylinder; 1 ml, 5 ml, and 10 ml glass pipettes•Absorbent paper towels•Automatic microtiter plate washer or laboratory wash bottle•Microtiter plate reader with 450 nm filter•Latex gloves, safety glasses, and other appropriate protective garments•Biohazard infectious waste containers•Safety pipeting devices for 1 ml or larger pipettes•TimerVI. Assay Techniques & Microtiter Plate WashingEfficient washing of the microtiter wells is a fundamental requirement of ELISA procedures, as thorough rinsing removes uncomplexed assay components and minimizes assay background. Using an automatic plate washer is recommended to enhance speed, efficiency, and well-to-well consistency. Manual plate washing can yieldequivalent results if performed carefully. Each Lenti-X p24 assay requires performing three, six-rinse cycles.IMPORTANT:•The Lenti-X p24 Assay contains reagent systems which are optimized and balanced for each kit lot. Do not interchange reagents from kits with different lot numbers. Do not interchange vial caps or stoppers eitherwithin or between kits.•Allow foil bags to warm to room temperature before opening. This avoids condensation on the inner surface of the bag, which may contribute to a deterioration of coated strips intended for future use.•Reagents should be dispensed with the tip of the micropipettes touching the side of the well at a point about mid-section. Follow manufacturer’s recommendations for automatic processors.•Always keep the upper surface of the microtiter strips free from excess fluid droplets. Reagents and buffer over-spill should be blotted dry on completion of the manipulation.•Do not allow the wells to completely dry during an assay.A. Automatic Plate WashingEach rinse cycle must consist of six consecutive washes. On completion of a rinse cycle, invert the plateor strips onto absorbent paper towels and tap firmly. Check for any residual wash buffer in the wells andblot the upper surface of the wells with a dry paper towel. In addition, automatic plate washers shouldmeet the following criteria:•All wells are completely aspirated.•All wells are completely filled (350 µl) during each rinse cycle.•Wash buffer is dispensed at a good flow rate.•The apparatus must be well maintained to prevent contamination from previous use. Perform cleaning procedures regularly, according to the manufacturer’s instructions.B. Manual Plate WashingFor manual plate washing, perform the following steps for each rinse cycle:1.Aspirate well contents using a vacuum line fitted with a trap to collect liquid.2.Fill all wells to the brim with wash buffer dispensed from either a multichannel pipettor or a squeeze-type laboratory wash bottle.3.Aspirate all wells.4.Repeat steps 2 and 3, five times.5.Invert the microtiter plate and tap firmly on absorbent paper towels.VII. Protocols: Lenti-X p24 Assay ProceduresIMPORTANT: Please read the entire protocol before starting. Detailed instructions are provided for thequantitative assay of p24.A. Protocol: Specimen Collection and StorageThe Lenti-X p24 Assay is intended for use with tissue culture supernatants. Specimens should be tested as soon as possible, but can also be stored frozen at –80°C, if necessary. Thoroughly mix thawed samplesbefore testing.B. Protocol: Wash Buffer PreparationPrepare 1X wash buffer by diluting 1 part Wash Buffer (20X) with 19 parts distilled or deionized water. If the kit will be utilized over a period greater than 4 weeks, then prepare only enough working strengthwash buffer for immediate needs. Each strip of 8-wells can be adequately washed with ~60 ml of working strength wash buffer.C. Protocol: Preparing Dilutions for the p24 Standard Curve (0.5 hr)NOTE: This note pertains to samples with High to Very High Levels of p24 (e.g., lentiviral supernatants generated using Lenti-X Packaging Single Shots). Samples containing high levels of p24 (i.e. >200pg/ml) must be diluted prior to assay in order to obtain accurate p24 values. Such samples may includelentiviral supernatants produced using Lenti-X Packaging Single Shots, which often require diluting 10–10,000-fold. We recommend making several serial 10-fold dilutions to generate at least one dilution in the range of the standard curve. Mix dilutions thoroughly before assaying or diluting them further, assay each sample in duplicate, and be sure to multiply each result by its dilution factor to determine the correct p24 value in the original sample.To test samples quantitatively and determine accurate virus titers, you will need to prepare a p24 standard curve (0–200 pg/ml). Examples of data collected for a typical standard curve and for test samples areshown in Table 1 and Figure 3. To prepare dilutions for the standard curve:1.Prepare a working strength p24 positive control stock solution by diluting 20 µl of the p24 Control(10 ng/ml) into 980 µl of fresh complete tissue culture medium (e.g., DMEM containing 10% FBS),for a 1:50 dilution. This will produce a 200 pg/ml stock solution.ing the 200 pg/ml stock and complete tissue culture medium as the diluent, make a series of fouradditional standard dilutions of 100, 50, 25, and 12.5 pg/ml. Dispense 500 µl of media into each offour labeled tubes. Add 500 µl of the 200 pg/ml stock into the 100 pg/ml tube, mix, and using a freshpipet tip, transfer 500 µl of this 100 pg/ml solution into the 50 pg/ml tube and mix. Repeat similartransfers for the 25 and 12.5 pg/ml tubes.D. Protocol: Assaying Your Lentiviral Supernatants (~4 hr)1.Allow all reagents to reach room temperature (18–25°C).2.Select a sufficient number of 8-well strips to accommodate all standards, test specimens, controls, andcomplete culture medium blanks (negative controls) in duplicate. Fit the strips into the holding frame.Label wells according to specimen identity using the letter/number cross reference system moldedinto the plastic frame.3.Dispense 20 µl of lysis buffer into each well.4.Dispense 200 µl of each standard curve dilution, supernatant sample, and culture medium intoappropriately labeled duplicate wells.5.Incubate at 37°C ± 1°C for 60 ± 5 min.6.Aspirate the contents of the wells, and wash the microtiter plate as described in Section VI.7.Dispense 100 µl of Anti-p24 (Biotin conjugate) detector antibody into each well.8.Incubate at 37°C ± 1°C for 60 ± 5 min.9.Aspirate the detector antibody from the wells, and wash the microtiter plate as described in Section VI.10.Dispense 100 µl of Streptavidin-HRP conjugate into each well.11.Incubate at room temperature (18–25°C) for 30 ± 5 min.12.Aspirate the conjugate from the wells, and wash the microtiter plate as described in Section VI.13.Without delay, dispense 100 µl of Substrate Solution into each well. A multichannel pipet should beused for best results.14.Protect the plate from direct light/sunlight, and incubate at room temperature (18–25°C) for 30 ± 2 min.15.Stop the reaction by adding 100 µl of Stop Solution to each well including the culture medium blanks.The blue solution should change to a uniform yellow color. Ensure that the undersides of the wells aredry and that there are no air bubbles in the well contents.16.Immediately after adding the Stop Solution, read the absorbance values at 450 nm using a microtiterplate reader blanked on the negative control well.Table 1. Sample data for standard curve.Absorbance (450 nm)Standard (pg/ml) A B Mean Sample (pg/ml)0 0.030 0.034 0.032 –12.5 0.145 0.155 0.150 –25 0.259 0.283 0.271 –50 0.501 0.531 0.516 –100 0.981 1.031 1.006 –200 1.800 1.820 1.810 –Test Sample 1 0.260 0.274 0.267 24.6Test Sample 2 0.611 0.637 0.624 61.0Figure 3. Lenti-X p24 Rapid Titer Kit standard curve. The curve was constructed using the p24 standards and results listed in Table 1. Samples were prepared and assayed as described.VIII. Interpretation of ResultsA. Assay ValidationThe following criteria are required for a valid assay:• A value of ≤0.10 for the negative control• A value of ≥0.60 for the 100 pg/ml standardB. Determining Your Virus TiterYour p24 values can be used to determine the relative virus titers of your packaging cell supernatants. Tocalibrate your virus production system and determine a relationship between p24 levels and infectivity, itmay be useful to determine the p24 levels of supernatants for which you have already measured the virustiter using an alternative method (i.e. determining infectious units based on expression of a fluorescentprotein or drug-selective marker).The following values and calculations may also be used to determine approximate titers, and are based onthe observation that each lentiviral particle (LP) contains approximately 2,000 molecules of p24: • 1 LP contains 8 x 10-5 pg of p24 (derived from (2000) x (24 x 103Da)/(6 x 1023)• 1 ng p24 is equivalent to ~1.25 x 107 LPs•For a typical lentivirus vector, there is 1 IFU for every 100–1,000 LPs•Therefore, a supernatant titer of 107IFU/ml ≈ 109–1010 LP/ml or 80–800 ng p24/ml Appendix A: Troubleshooting Guide1.p24 values determined using assays from different manufacturers or different methods may not be usedinterchangeably.2.The assay cannot be used to quantitate samples having p24 values greater than the highest value on the p24standard curve, unless the samples are diluted sufficiently. See Section VII for more information.3.The performance characteristics have not been established for any matrices other than tissue culture media.4.Signs of reagent deterioration are as follows:•The kit fails to meet the required criteria for a valid test (see Section VIII.A).•Reagents become visibly cloudy or contain a precipitate.NOTE: The 20X Wash Buffer normally develops a crystalline precipitate during storage at 2–8°C. Thiswill dissolve upon warming at 37°C.•The TMB Substrate solution becomes dark blue in color. This is likely caused by chemical contamination of the substrate solution.Contact UsCustomer Service/Ordering Technical Supporttel: 800.662.2566 (toll-free) tel: 800.662.2566 (toll-free)fax: 800.424.1350 (toll-free) fax: 800.424.1350 (toll-free)web: web: e-mail: **********************e-mail: ********************Notice to PurchaserOur products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without prior written approval of Takara Bio USA, Inc.Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by such statements.©2016 Takara Bio Inc. All Rights Reserved.All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at .This document has been reviewed and approved by the Quality Department.。
莫比特x s15d双摄像头手册说明书
MOBOTIX S15DFlexMount Dual CameraMOBOTIX AdvantagesExtremley high-sensitive 5-megapixel sensors • All relevant events are automatically saved • Highly secure as motion sensors detect even the smallest movements and reduce the number of false alarms • Cost-effective as fewer cameras are needed thanks to HiRes panorama view • Maintenance-free with no heaters, fans or moving parts • Can be expanded thanks to encrypted two-wire bus (MxBus) • Synchronization via GPS satellite means data available in real time • Weatherproof from -30°C to 60°C (-22°F to 140°F) • No licensing or software fees, free-of-charge updatesT e c h n i c a l s p e c i fic a t i o n s s u b j e c t t o c h a n g e w i t h o u t n o t i ceFor more information on the entire range of MOBOTIX accessories and additional information on the S15D, such as prices, manuals, video management software for computers and iOS devices, etc., please go to > Products. Or - if you would like to talk to us on the phone to get advice with our products, please call +49 06302 9816-103.Camera Model S15D-Sec Lenses, Sensors (Optical, Sensor Modules And BlockFlexMount Sensor Modules)Hemispheric 12 mm (180° x 160°)L12Super Wide-Angle 25 mm (82° x 61°)L25Wide-Angle 38 mm (55° x 41°)L38Wide-Angle 51 mm (40° x 30°)L51Tele 76 mm (27° x 20°)L76Tele 160 mm (13° x 10°)L160Tele 320 mm (7° x 5°)BlockFlexMount only CSVario 28 – 63 mm (28° – 58°)BlockFlexMount only Image sensor with individual exposure zones (B/W sensor modules also available with integrated Long Pass Filter)Color, B/W (any combination)Sensor sensitivity in lux at 1/60 s /1 sColor: 0,25/0,013 B/W: 0,005/0,0025Image sensor resolution (each color or B/W sensor)5MP (2592 x 1944) Thermal Sensor Modules For S15D see pages 7 to 9Hardware FunctionsProtection class (camera body)IP65 Temperature range -30 to +60°C/-22 to +140°F•Temporary internal DVR64 MB Internal DVR, ex works 4 GB (MicroSD) Microphone (in sensor modules)/speaker•/–Passive infrared sensor (PIR)–Integrated temperature sensor•Shock detector•Power consumption (typical) with one/two sensor module(s, not with thermal sensor modules< 4,5 watts/< 5 watts Variable PoE class (factory default: class 3, with thermal sensor module class 3 always necessary) 2 or 3 Image Formats, Frame Rates, Image StorageMaximum image format (per sensor)5MP (2592 x 1944) Maximum frame rate (MxPEG, max. resolution)10 fps (5MP) CIF images with 4 GB MicroSD DVR250,000VGA images with 4 GB MicroSD DVR125,000HD images with 4 GB MicroSD DVR40,000 QXGA images with 4 GB MicroSD DVR20,000• available ex works– not availableCamera Model S15D-Sec General FuncrionsDigital zoom (continous) with panning•Codecs: Motion-JPEG/MxPEG/H.264 for SIP•/•/•Programmable exposure zones•Snapshot recording (pre/post-alarm images)50 Terabyte ring buffer storage (internal/network)•Continous recording with sound (0.2 to 30 fps)•Event recording with sound•Time and event control/flexible event logic•/•Weekly schedules/holidays•Web functionality (FTP, email)•Playback/QuadView and MultiView•/•Bidirectional sound in browser – extra speaker needed*•Logo generator, animated•Master/Slave arming•Several scheduled privacy zones•Customized voice messages – extra speaker needed*•VoIP telephony (audio/video, alert) – extra speaker needed*•Remote alarm notification (network message)•Signal inputs/outputs and RS232 via MX-232-IO-Box Programming interface (HTTP API)•Security Features•(HTTPS/SL, IP-based access control, IEEE 802.1X network authentification)Video AnalysisVideo motion detector•MxAnalytics–MxActivitySensor•Video Management SoftwareMxEasy (Windows/Mac OS X)•MxControlCenter (Windows)•MOBOTIX App (iOS)•* Niote: No integrated microphone in a thermal sensor moduleS15D Core (Basis odul )1.11.111.8 1.71.61.31.41.101.91.21.5M.6M.3M.2M.1M.7M.8M.5M.4M.10M.9(Excerpt from the technical documentation: > Support > Manuals)54.0130.039.0Ø5.517.3100.0100.08.5Ø7.033.510.0115.016.0Ø19.030.0110.0106.047.515.037.05.019.124.544.0103.0Ø5.5Ø4.0BottomSideTopFrontExample with L12 Sensor, ceiling mount.Each extension gives you 40mm more space.You can use more than one on every sensor.Ø45.0Ø45.0Ø45.0+ 2x 40m m = 85+ 40m m = 45m a x 5m m(Excerpt from technical drawings and 3D views: > Support > Image Database)Delivered Parts S15D Core (Basis Module)Pressure compen-sationBase plateRLLEDs MicroSD cardKeysTriple cable retainerBayonet catch S15D housing cover Sensor modulesMiniUSBEthernet patch cableEthernet installation cable or MxBus, microphone, speakerMOBOTIX-RJ45 or MxBus, microphone,speakerLSA+ terminal Ethernet Installationcable Screw terminals MxBus, microphone,speaker(S15D – Camera housing and connectors, excerpt from the technical documenation)Thermal Sensor Modules For The S15DThe world’s first flexible dual thermal cameraThe thermal sensor modules measure the thermal radiation ofobjects, so that they can function in absolute darkness. Togetherwith the MxActivitySensor, they can reliably detect motion inimages at night. Only changes in position trigger a signal. Objectsmoving on the spot do not trigger a signal. The thermal sensormodules also have an advantage during the day since they candetect moving objects in shadows, semi-darkness, smoke, orbehind bushes.The MOBOTIX thermal sensor modules are designed for around-the-clock operation in industrial conditions and are certified asweatherproof according to IP66. Just like for the daylight modules,there are different focal lengths available for the thermal modules:•MX-SM-Thermal-L43 with a horizontal image angle of 45°•MX-SM-Thermal-L65 with a horizontal image angle of 25°•MX-SM-Thermal-L135 with a horizontal image angle of 17°The S15D with thermal sensor module(s) in a weatherproof aluminum housing is the world’s first flexible dual thermal camera. This is because, as is typical for the S15D, the new thermal sensor modules are also flexibly connected with the familiar, max. two-meter-long sensor cables to the camera housing, which makes efficient installations and customer-specific special installations very easy.Figure: One camera secures two surveillance areas, for example 90° around a corner, indoor and outdoor areas, two areas with different focal lengths, left/right side.The following thermal sensor module combinations are possible with the S15D:A. Mixed operation (1 x thermal sensor module, 1 x 5 MP sensor module):The advantages of an S15D with a thermal sensor module and simultaneous daylight sensor lie in theandinthehoursandduringtwilightdayofcombinationbothimages:5-megapixelbrilliantimagesreliable motion detection at night.B. Dual thermal operation (2 x thermal sensor module – only possible with the S15D, not with the M15D-Thermal):Two thermal images of two different image areas with just one cameraC. Single thermal operation (1 x thermal sensor module):One thermal image, thermal sensor module with flexible mountingB. C.A.Retrofit or upgrade with thermal technology is possible at any timeIn combination with the camera firmware version 4.2.1.43 or higher, every S15D can operate with thermal sensor modules and be converted into a high-end thermographic camera.Maximum spacing between surveillance camera and detected object with the thermal sensor modules L43 to L135: Recognition criteria according to EN 50132-7L43 / 45°L65 / 25°L135 / 17°Monitoring of humans52 m95 m144 mDetection of humans26 m47 m72 mMonitoring of cars150 m275 m400 mDetection of cars58 m140 m200 mHighly sensitive thermal sensor with NETD typically 50 mK (visualizes temperature differences starting at 0.05°C/0.09 °F)Unlike cameras with 5 MP image sensors, one of the decisive quality criteria for a thermal camera is not the image resolution, measured in pixels, but rather the camera’s ability to capture the slightest differences in temperatureand to produce an image that displays these differences in colors. The sensitivity of a thermal sensor is measuredin millikelvin by the NETD, or Noise Equivalent Temperature Difference. MOBOTIX thermal cameras offer a sensorvalue of 50 mK, which places them in the top range.Warehouse:Thanks to an NETD of 50 mK,the MOBOTIX thermal image(left) shows more details thana less powerful thermographiccamera with an NETD of 100mK (right)T echnical data - Thermal sensor modules for S15DModel versionsMX-SM-Thermal-L43/L65/L135, dual operation with an additional thermal sensor or MX sensor module (5 MP) possible on the S15D Lens options for thermal image sensor L43: 45°, L65: 25°, L135: 17° (horizontal image angle)Sensitivity - thermal image sensor NETD typically 50 mK (equivalent to 0.05 °C/0.09 °F), < 79 mK Image sensor for thermal image sensor Uncooled microbolometer with 336 x 252 pixelsTemperature measurement range -40 °C to +550 °C/-40 °F to +1022 °F (temperature of objects to be detected)Spectral range7.5 to 13.5 μmMaximum image size for thermal image sensorScalable up to 2592 x 1944 (5MP), automatically scaled to the image size of the MX sensor module with dual imagesMaximum frame rate for thermal image sensor9 fps (the camera’s overall frame rate is reduced to a maximum of 9 fps when an MX sensor module and a thermal image sensor are displayed simultaneously)Software functions for thermal image sensor (certain features only available with firmware version 4.2.1.43 and higher)Optional off-color or black and white image, image mirroring, obscure image area, PTZ commands (pan, tilt, zoom), text and logo options, show event/action symbols, level displays in bars or diagrams, temperature control windowPower consumption for S15D with one/two thermal sensor module(s)Typically 1.5 W per thermal sensor module; however, can only be used together with an S15D camera housing (versions A to C):A. Mixed operation (1 x thermal, 1 x optical): typically 6.5 W (7.5 W possible over the short term)B. Dual thermal operation (2 x thermal): typically 7 W (8 W possible over the short term)C. Single thermal operation (1 x thermal): typically 5.5 W (6.5 W possible over the short term)Operating conditions IP66, -30 °C to +60 °CMaterialModule housing: black anodized aluminum; pressure plate: V2A stainless steel; lens and protective glass lens: germaniumWeight / length / installationdimensions Thermal sensor modulesWeight: < 330 g (one thermal sensor module without sensor cable); total length: 78 mm; diameter of front panel: 57 mm; diameter of stainless steel pressure plate: 63 mm; bore diameter: 48 – 53 mm; maximum wall thickness for installation: 14 mm; alternativemounting with six screw threads on the side of the module for M4 screws, 4 mm thread depthDelivered partsThermal sensor module, 3 mm Allen wrench used to install the pressure plate, QuickInstall guide - the S15D camera housing (S15D-FlexMount Core) and sensor cable must be ordered separately!NOTE: An S15D with one or two thermal sensor modules always requires PoE class 3 (factory default).Attention – Special Export Regulations For Thermal Cameras Apply!Cameras with thermographic image sensors (“thermal cameras”) are subject to the specialexport regulations of the U.S.A. and the ITAR (International Traffic in Arms Regulation):• According to the currently applicable export regulations of the U.S.A. and the ITAR, cameras with thermographic image sensors or parts thereof must not be exported to countries embargoed by the U.S.A. or the ITAR. At present, this applies to the following countries: Syria, Iran, Cuba, North Korea and Sudan. The same export ban applies to all persons and institutions listed in “The Denied Persons List” (see , “Policy Guidance > Lists of Parties of Concern”).• Under no circumstances can the camera itself or its thermographic image sensors be used in the design, the development or in the production of nuclear, biological or chemical weapons or in the weapons themselves.。
慢病毒包装操作说明
Clontech-Lenti-X™ Lentiviral Expression Systems User ManualProtocol No.PT5135-1慢病毒包装操作说明A.用Lenti-X HTX Packaging System生产慢病毒悬浮物为了获得最高效价的病毒悬液，用Lenti-X 293T细胞系，严格尊守以下说明，尤其尊守（1）培养体系和培养量（2）DNA的量和转染质量（3）无四环素血清（4）孵育时间。
所有的Xfect™转染成份，量和条件最好用Lenti-XVectors，Lenti-XHTX包装混合物，Lenti-X293T细胞。
用10cm组织培养板并确保血清无四环素，四环素污染的血清对表达包装成份是有害的。
所有的实验步骤均在无菌组织培养器血中完成。
包装病毒需要有微生物安全等级2的生物安全柜中进行，注意重组的假性慢病毒包装颗粒能够感染人。
61.转染24小时前，在10cm培养板接种4-5×10个293T细胞，添加10ml的生长培养基。
在37℃，5%CO2℃条件下过夜。
在进行第7步前确保培养血有80-90%的覆盖率。
2.充分混均Xfect Polymer。
3.每个转染样品需准备两个离心管，按顺序添加如下试剂Tube 1(Plasmid DNA) Tube 2(Polymer)557µlXfectReaction Buffer592.5µl Xfect ReactionBuffer36µl Lenti-X HTX Packaging Mix7.5µl Xfect Polymer7µl Lenti-X Vector DNA(1µg/µl)600µl 总量600µl 总量注意：Xfect Polymer不要在室温下搁置长于30min4.充分混均每个管5.把Tube2添加到Tube1中，中速涡旋10秒。
pLVX-AcGFP1-N1
(122111)pL VX-AcGFP 1-N1 Vector Information PT3994-5Catalog Nos. 632154pL VX-AcGFP1-N1 Vector Map and Multiple Cloning Site (MCS).DescriptionpLVX-AcGFP1-N1 is an HIV-1-based, lentiviral expression vector that allows you to express your gene of interest fused to AcGFP1, a green fl uorescent protein derived from Aequorea coerulescens . Genes cloned into the multiple cloning site (MCS), located upstream of the AcGFP1 coding sequence, are expressed as N -terminal fusions of the AcGFP1 protein. Expression of the fusion protein is driven by the constitutively active human cytomegalovirus immediate early promoter (P CMV IE ) located just upstream of the MCS. Lentiviral particles derived from the vector allow the expression of AcGFP1 fusion proteins in virtually any cell type, including primary cells. T he unmodifi ed vector expresses AcGFP1, and may be used to produce marker virus to optimize infection protocols.pLVX-AcGFP1-N1 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RN A (1), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RN A out of the nucleus (2). Finally, pLVX-AcGFP1-N 1 also contains a central polypurine tract (cPPT) element that increases nuclear importation of the viral genome during target cell infection, resulting in improved vector integration and more effi cient transduction (3).Puror2816CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCABamHISmaIXmaIApaIEcoRIBstBI2870CCG GTC United States/Canada800.662.2566Asia Pacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.543.6116Clontech Laboratories, Inc.A T akara Bio Company 1290 T erra Bella Ave.Mountain View, CA 94043Technical Support (US)E-mail: tech@ Clontech Laboratories, Inc. Protocol No. PT3994-52 Version No. 122111In addition to lentiviral elements, pLVX-AcGFP 1-N1 contains a puromycin resistance gene (Puro r ) under the control of the murine phosphoglycerate kinase (PGK) promoter (P PGK ) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Amp r ) for propagation and selection in epLVX-AcGFP1-N1 constitutively expresses your gene of interest from P CMV IE when transduced into target cells. Before the vector can be transduced into cells, however, it must be transfected into 293T packaging cells with our Lenti-X™ HT Packaging System (Cat. Nos. 632160 and 632161). T his packaging system allows you to safely produce high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (4).Location of Features • 5' LTR: 1–635• PBS (primer binding site): 636–653• Ψ (packaging signal): 685–822• RRE (Rev-response element): 1303–1536• cPPT(central polypurine tract): 2028–2151• P CMV IE (human cytomegalovirus immediate early promoter): 2185–2788• MCS (multiple cloning site): 2816–2868• AcGFP1 (Aequorea coerulescen s green fl uorescent protein): 2876–3592• P PGK (phosphoglycerate kinase promoter): 3614–4122• Puro r (puromycin resistance gene ): 4143–4742• WPRE (woodchuck posttranscriptional regulatory element): 4756–5347• 3' LTR: 5551–6187• pUC origin of replication: 6657–7327 (complementary)• Amp r (ampicillin resistance gene; β-lactamase): 7472–8468 (complementary)Selection of Stable T ransfectants• Selectable marker: plasmid confers resistance to puromycin.Propagation in E. coli• Suitable host strains: DH5α, DH10B and other general purpose strains.• Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.• E. coli replication origin: pUC • Copy number: highNotes:The attached sequence fi le has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. T his vector has not been completely sequence.The viral supernatants produced by this lentiviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and handling of recombinant lentivirus. Appropriate NIH, regional, and institutional guidelines apply.References1. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892.2. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.3. Zennou, V. et al. (2000) Cell 101(2):173–185.4. Wu, X. et al. (2000) Mol. T her. 2(1):47–55.Notice to PurchaserClontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modifi ed for resale, or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories, Inc.Your use of this product is also subject to compliance with the licensing requirements described on the product´s web page at. It is your responsibility to review, understand and adhere to any restrictions imposed by these statements. Clontech and the Clontech logo are trademarks of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. Clontech is a T akara Bio Company. ©2011 Clontech Laboratories, Inc.This document has been reviewed and approved by the Clontech Quality Assurance Department.Protocol No. PT3994-5 Clontech Laboratories, Inc. Version No. 122111 3。
Siemens 6XV1830-3EH10 产品说明书
● halogen-free
No
● silicon-free
Yes
Standards, specifications, approvals UL/ETL listing / 300 V Rating UL/ETL style / 600 V Rating Certificate of suitability ● CE marking ● RoHS conformity Marine classification association ● American Bureau of Shipping Europe Ltd. (ABS) ● Bureau Veritas (BV) ● Det Norske Veritas (DNV) ● Germanische Lloyd (GL) ● Lloyds Register of Shipping (LRS) ● Nippon Kaiji Kyokai (NK) ● Polski Rejestr Statkow (PRS)
● to mineral oil ● to grease ● to water Radiological resistance / to UV radiation
Limited segment length (see manual for PROFIBUS networks) flame resistant according to IEC 60332-1-2
6XV1830-3EH10 Page 3/3
08/08/2017
Subject to change without notice © Copyright Siemens
Suitability for use Cable designation
EGFR和PD-L1双靶向嵌合抗原受体构建及表达
•论著•EGFR和PD-L1双靶向嵌合抗原受体构建及表达李姝萍王小珏杨斌王和林闫卓红易玲韦攀健金鑫郝建清张洪涛【摘要】目的构建肿瘤相关抗原表皮生长因子受体特异性嵌合抗原受体(EGFR-CAR)和程序性死亡受体-配体1(PD-L1)抗体双修饰慢病毒载体表达系统’方法人PD-Ll-Fc蛋白免疫BALB/c小鼠，经细胞融合、亚克隆筛选高分泌PD丄1特异性抗体的稳定杂交瘤，酶联免疫吸附试验(ELISA)和Western blot检测抗体特异性.流式细胞术(FACS)鉴定对PD-1配受体封阻性能，Fortebio测定抗体亲和力,抗体全长测序，经保留鼠源CRD1,CRD2和CRD3人源化改造后构建单链抗体(single-chain variable fragment,scFv):人EGFR单克隆抗体杂交瘤系，经5'RACE技术扩增其轻链和重链可变区(VL和VH)基因，构建scFv,克隆至真核载体pcDNA3」表达鉴定。
基因合成EGFR-CAR(引人CD137协同信号胞内功能域)与PD-Ll-scFv借助2A序列连接，克隆入慢病毒pLVX-EFla-IRES-ZsGreenl表达载体.使用Lenti-X Packaging Single Shots(VSV-G)共同转染293T细胞，获得包装病毒，感染293V细胞，FACS测定CAR膜表达，ELISA检测CAR 感染293V细胞培养上清中PD-Ll-scFv表达情况.转染激活人外周血T细胞，验证CAR膜表达。
结果获得PD-L1抗体11E3.具备高度配受体封阻性能，经人源化改造后，亲和力稳定(2.67x IO'10mol/L),EGFR-scFv获得有效表达：进一步构建了EGFR-CAR和PD-L1双修饰慢病毒分泌型CAR(CTC0537-1)及膜表达型CAR(CTC0537-2),其病毒感染293V细胞阳性率为10%。
CTZ0431-1感染293V细胞后，细胞膜表面表达EGFR-scFv,检测培养上清存在PD-Ll-ScFv；CTZ0431-2感染293V细胞后，细胞膜表面EGFR-scFv和PD-Ll-scFv有效表达，双表达病毒感染活化T细胞的CAR表达率为39.3%：结论成功构建了EGFR-CAR和PD-Ll-scFv双表达慢病毒载体，EGFR-CAR中度结合亲和力，此为EGFR靶向和PD-L1抗体双修饰CAR-T细胞的实体瘤治疗研究提供了关键工具。
Lenti-X HTX 慢病毒包装系统操作流程(clontech)
Lenti-X™ HTX 高效慢病毒包装系统操作一览PT5135-2目录重要 (1)存储&处理 (1)转染操作 (2)重要：以下操作是针对Lenti-X 载体，Lenti-X HTX 高效包装系统和Lenti-X 293T 细胞系(Cat. No. 632180)的优化操作。
转染操作需在100 mm 培养板中进行，转染培养基和收集病毒的培养基中需使用Tet System Approved FBS (无四环素污染)。
以下所有操作需在无菌环境下进行，需要使用生物安全等级为2的仪器设备进行病毒操作并做好自身的防护措施。
存储&处理：●使用前，室温解冻Xfect™ Polymer (100 μg/μl)。
解冻后，将Xfect Polymer 置于4°C保存，最多12 months。
●使用前，室温解冻Xfect Reaction Buffe。
解冻后摇匀，将Xfect Polymer 置于4°C保存，最多12 months。
●使用完Xfect Polymer后，盖紧管盖，放回带有干燥剂的锡箔袋。
Figure 1. Lenti-X 293T细胞最优转染密度（左图）和转染后细胞检测时间（右图），转染筛选标记是ZsGreen 绿色荧光蛋白。
转染操作（100 mm板）：1.转染前约24 hr，以4–5 x 10e6Lenti-X 293T细胞/100 mm板的密度接种10 ml细胞，37°C、5% CO2 培养过夜。
转染时的细胞融合度应该为80–90%。
2. 漩涡混匀fect Polymer。
3. 对于每个转染样本，准备2个离心管，按照以下顺序混匀试剂：注意 Xfect Polymer预混液室温放置不要超过30min。
4. 漩涡混匀各管混合物。
5. 将Xfect Polymer预混液和DNA预混液以适中的速度漩涡10sec。
6. 室温孵育10 min，以形成便Xfect/DNA复合物。
Polaris Series 6000i 8x8到192x192光路切换器说明书
DIRECTLIGHT TECHnoLoGyThe Series 6000i 8x8 to 192x192 switch leverages Polatis’ patented, highly reliable piezoelectric DirectLight beam-steering technology that sets the industry standard for lowest optical loss and highest optical performance. Polatis' beam-steering technology can be switched without light being present on the fiber and can also switch bi-directional signals. This allows operators to pre-provision paths, as well as switch intermittent and variable-power test signals, over lit or dark fiber. Ultra-high performance is now available for the 6000i-Ultra in matrix sizes up to 96x96 with <1.0dB max insertion loss.SDn EnABLED WITH USER FRIEnDLy InTERFACESPolatis offers a full complement of Software Defined Networking (SDN) interfaces including NETCONF, and RESTCONF. Optical switching with SDN allows infrastructure vendors and system test operators to dynamically and cost effectively setup, monitor and operate cloud-based test configurations. Polatis works closely with leading SDN companies and research organizations to provide leading edge SDN solutions. In addition, Polatis also offers traditional SNMP , TL1, GPIB, and SCPI command languages that allow for seamless integration with test equipment controller systems. Each switch also has a user-friendly secure web browser GUI interface that can be used to provision, monitor, and control the switch and the switch software can be easily upgraded in the field without affecting in-service switch operations.FLEXIBLE SWITCH MATRIX SIZE oPTIonSThe Series 6000i switch is available in matrix sizes from 8x8 to 192x192 in a variety of matrix configurations, including symmetric (NxN), asymmetric (MxN), and (NxCC) customer configurable, to meet a broad range of testing applications. Polatis offers two different versions of the Series 6000i: the high-performance 8x8 to 96x96 Ultra, and the high-port count 108x108 to 192x192 6000i. The 6000i’s large matrix size,combined with its low loss characteristics, allows for building multistage scalable switch solutions that can grow to interconnect thousands of ports.InTEGRATED FEATURES FoR TEST LAB APPLICATIonSPolatis Series 6000i switches can be customized to incorporate a variety of passive and active components to suit individual customer testing needs. These include options for integrated Optical Power Monitors (OPMs) and optical taps on every connection. The power monitoring can be used to provide Variable Optical Attenuation (VOA) on every connection and the taps can used for signal monitoring or multicast. In addition, Polatis instrument grade switches have a unique user-programmable shutter function that can be used to create single or repeated fiber breaks on any number of switchconnections for network stress testing.SINGLE MODE INSTRUMENT OPTICAL SwITCh FROM 8x8 TO 192x192 PORTS6000iInstrumentOptical Matrix SwitchAchieve More with Optical Switching ™The Polatis Series 6000i Instrument optical switch is a high-performance, fully non-blocking all-optical matrix switch available in sizes from 8x8 up to 192x192. It is designed to meet the highest performance needs of the most demanding test and measurement applications with exceptionally low optical loss, superior connection stability and repeatability in a compact form factor. with support of Software-Defined Networks (SDNs) via embedded NETCONF and RESTCONF control interfaces, the Series 6000i interfaces directly with cutting edge cloud-based network and infrastructure testing applications. The Series 6000i is based on Polatis’ patented DirectLight ® optical switching technology that has been proven in the most challenging defense, data center and telecom applications and is exclusively used by major network equipment manufacturers to automate testing of optical components and subsystems.Series 6000 Ultra 32x32 Optical SwitchSeries 6000 192x192 Optical SwitchKEy FEATURESUltra-high performance now available for the 6000i Ultra in sizes up to 32x32with <1.0dB and 96x96 with <1.2dB max insertion loss• Non-blocking matrix switch sizes from 8x8 to 192x192• Ultra-low insertion loss and superior optical specifications • Exceptional optical stability and repeatability • Dark fiber all-band single mode connectivity • Fully bidirectional optics• Available in NxN, MxN single-sided,and customer configurable (NxCC)any-to-any port configurations • Protocol and bit-rate agnostic up to 400Gbs and beyond • Optional Optical Power Monitoring (OPMs) with user configurable optical power alarms • Optional Variable Optical Attenuation (VOAs) on every switch connection • Programmable port shutter for fiber break simulation • SDN enabled with NETCONF and RESTCONF command interfaces • Configurable interface options with SNMP , TL1, and SCPI control languages • Built-in user-friendly Web GUI • High reliability distributed architecture • High density switching in a compact chassis • Eco-friendly energy efficiency chassis• Supports RADIUS secure user access protocols。
Lenti-X iDimerize Inducible Homodimer System说明书
Product Components List Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: *****************United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan+81.(0)77.543.6116 Page 1 of 1Last update: 10/18/2011 Lenti-X™ iDimerize™ Inducible Homodimer SystemCatalog No. Amount 635072 each DescriptionThe Lenti-X iDimerize Inducible Homodimer System lets you control the homodimerization of a protein of interest in live cells by adding a small molecule, the membrane-permeant compound B/B Homodimerizer, to the culture medium. The system includes the Lenti-X iDimerize Inducible Homodimer Vector Set 1, which contains three lentiviral vectors encoding dimerization tags (DmrB), as well as subcellular localization tags that can be easily fused to your protein of interest. In combination with the Lenti-X HTX Packaging System (Cat. Nos. 631247 & 631249), the optimized Lenti-X constructs allow for packaging and delivery of high-titer lentivirus to the widest range of cell types. Using this system, the activity and/or localization of the resulting chimeric protein can be controlled by the addition of B/B Homodimerizer, which is required for homodimerization and is also included.Package ContentsLenti-X iDimerize Inducible Homodimer Vector Set 1 (Cat. No. 635073; Not sold separately) >> View Components 500 μl B/B Homodimerizer (0.5 mM; Cat. No. 635060) >> View ComponentsFor storage conditions, please see the Certificate of Analysis supplied with each component.Product DocumentsDocuments for Clontech products are available for download at /manuals The following documents apply to this product:Lenti-X iDimerize Inducible Homodimer System User ManualNotice to PurchaserClontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories, Inc.Your use of this p roduct is also subject to compliance with any applicable licensing requirements described on the product’s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by such statements.Clontech, the Clontech logo, iDimerize, and Lenti-X are trademarks of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. Clontech is a Takara Bio Company. ©2011 Clontech Laboratories, Inc.This document has been reviewed and approved by the Clontech Quality Assurance Department.。
Kinetix 6200和6500服务变速器多轴用户手册说明书
Instrucciones de instalaciónJuegos de conectores de perfil bajo para señales de E/S, seguridad y retroalimentación auxiliarNúmero de catálogo 2090-K6CK-D44MAcerca de los juegos de conectores de perfil bajoEste juego de conectores de 44 pines ofrece puntos de terminación para E/S, conexiones de retroalimentación auxiliar y de seguridad para el conector IOD de las unidades Kinetix® 6200 y Kinetix 6500.El juego también incluye un puente habilitador de movimiento que se puede instalar cuando no se desea activar la funcionalidad de desconexión de par segura de los módulos de control 2094-xx02x-M0x-S0. El puente no es apto para los módulos de control 2094-xx02x-M0x-S1. Estos documentos contienen información adicional sobre el cableado de E/S, la seguridad y las conexiones de retroalimentación auxiliar para las unidades de servovariadores Kinetix 6200 y Kinetix 6500.2 Juego de conectores de perfil bajo para señales de E/S, seguridad y retroalimentación auxiliarRockwell Automation Publicación 2090-IN021D-ES-P - Enero 2015Cómo instalar el juegos de conectores de perfil bajoATENCIÓN: Este juego de conectores contiene piezas delicadas a descargas electrostáticas (ESD),que pueden sufrir daños si no se cumplen los procedimientos de control de ESD. Si no conoce los procedimientos de control de descargas electrostáticas (ESD), consulte la publicación n.º8000-4.5.2, Guarding Against Electrostatic Damage, o bien, cualquier otro manual de protección contra ESD que corresponda.1.Coloque el blindaje del cable expuesto en el canal.2.Conecte los cables a los terminales.3.Coloque la abrazadera de blindaje sobre el blindaje expuesto.4.Apriete los tornillos con un par de 0.4 N•m (3.5lb•pulg.).abrazadera para que los cables pequeños queden bien sujetos.Utilice abrazaderas de blindaje (3) para maximizar el contacto con el blindaje del cable en conexiones equipotenciales de alta frecuencia.Utilice sujetacables (4) para reducir la fatiga mecánica.Juego de conectores de perfil bajo para señales de E/S, seguridad y retroalimentación auxiliar 3Rockwell Automation Publicación 2090-IN021D-ES-P - Enero 2015Datos de los conectores(1)Los indicadores que se encuentran entre paréntesis hacen referencia al relé de seguridad Guardmaster® MSR57P y a los terminales de opción de seguridad serie 750 de PowerFlex®.(2)Utilice este suministro para alimentar la entrada de seguridad de 24 V (SPWR/SCOM). No conecte esta entrada de 24 V a ningún dispositivo de seguridad externo. Estos pines no son aptos para los módulos de control 2094-xx 02x -M0x S1.Instalación de puente habilitador de movimiento (aplica a los módulos de control 2094-xx 02x -M0x -S0).La numeración de pines del juego corresponde al conector IOD. Se asignan varios terminales a los pines 27, 28, 39 y 40 para que puedan adaptarse conexiones adicionales.Publicación 2090-IN021D-ES-P - Enero 2015© 2015 Rockwell Automation, Inc. Todos los derechos reservados. Impreso en EE.UU.Allen-Bradley, CompactLogix, Kinetix, MicroLogix, Rockwell Software y Rockwell Automation son marcas comerciales de Rockwell Automation, Inc.Las marcas comerciales que no pertenecen a Rockwell Automation son propiedad de sus respectivas empresas.Rockwell Automation mantiene información ambiental actualizada sobre los productos en su sitio web en/rockwellautomation/about-us/sustainability-ethics/product-environmental-compliance.page .Datos de los conectores (continuación)(1)Los indicadores que se encuentran entre paréntesis hacen referencia al relé de seguridad Guardmaster MSR57P y a los terminales de opción de seguridad serie 750 de PowerFlex.(2)Utilice estas señales como fuente de 24 VCC para operar las entradas digitales (máximo de 50 mA por entrada).Instalación de puente habilitador de movimiento (aplica a los módulos de control 2094-xx 02x -M0x -S0).La numeración de pines del juego corresponde al conector IOD. Se asignan varios terminales a los pines 27, 28, 39 y 40 para que puedan adaptarse conexiones adicionales.。
炉子产品说明书
Serie 6, Forno da incasso, 60 x 60cm, AcciaioHBA257BS0Accessori integrati1 x Griglia combinata, 1 x Leccarda universale smaltataAccessori opzionaliHEZ538000 Guide telescopiche clip a 1 livello, HEZ538200 Guide telescopiche a 2 livelli, HEZ538S00 Guide telescop. a 2 livelli+1 guida clip, HEZ625071 Teglia per grigliare adatta a pirolisi,HEZ633001 Coperchio per tegame professionale, HEZ633070 Tegame professionale, HEZ634000 Griglia combinata 455x375x31 mm (LxPxA), HEZ636000 Leccarda in vetro 455x364x30 mm (LxPxA), HEZ638000 Guide telescopiche clip a 1 livello, HEZ638200 Set griglie telesc.2 liv.ad pl, HEZ638300 Set griglie telesc.3 liv.ad pl, HEZ660050 Accessory, HEZ664000 Griglia combinata 455x375x59 mm (LxPxA), HEZ915003 Pirofila in vetro con coperchio 5,4 l., HEZG0AS00 Cavo di collegamento 3m, HEZ317000 Teglia per pizza, HEZ327000 Pietra per pane e pizza, HEZ333001 Coperchio per leccarda extra profonda, HEZ530000 2 leccarde slim 455x188x39 mm (LxPxA), HEZ531000 Leccarda bassa 455x375x30 mm (LxPxA), HEZ531010 Leccarda antiaderen 455x375x30mm (LxPxA), HEZ532000 Leccarda profonda 455x375x38 mm (LxPxA), HEZ532010 Leccarda antiaderen 455x400x38mm (LxPxA),HEZ533000 Leccarda profonda 455x375x81 mm (LxPxA)Forno da incasso di moderno ed elegante design con programmi automatici di cottura: per preparare piatti perfetti.• Programmi automatici di cottura: cucinare sarà semplicissimo grazie ai programmi con impostazioni già preselezionate.• Comode manopole a scomparsa push-pull: per una pulizia piùsemplice del panello frontale.• EcoClean Direct: facile pulizia grazie a un rivestimento che dissolve il grasso durante la cottura.Dati tecniciTipologia costruttiva del prodotto: .....................................Da incasso Sistema di pulizia: ....Idrolisi, 3 pannelli catalitici, 3 pannelli catalitici Dimensioni del vano per l'installazione (AxLxP): 585-595 x 560-568 x 550 mmDimensioni (AxLxP): ............................................595 x 594 x 548 mm Dimensioni del prodotto imballato (AxLxP): .......675 x 660 x 690 mm Materiale del cruscotto: ..............................................................vetro Materiale porta: ..........................................................................vetro Peso netto: ..............................................................................34.1 kg Volume utile: .................................................................................71 l Metodo di cottura: .Grill a superficie grande, Aria calda delicata, aria calda, Riscaldamento statico, Funzione pizza, riscaldamento inferiore, grill ventilatoMateriale della cavità: .................................................................Altro Regolazione della temperatura: ..........................................Meccanico Numero di luci interne: (1)Lunghezza del cavo di alimentazione elettrica: .....................120.0 cm Codice EAN: (4242005056309)Numero di vani - (2010/30/CE): (1)Classe di efficienza energetica: .........................................................A Energy consumption per cycle conventional (2010/30/EC): ........0.97 kWh/cycleEnergy consumption per cycle forced air convection (2010/30/EC):0.81 kWh/cycleIndice di efficienza energetica (2010/30/CE): ..........................95.3 % Potenza: ..................................................................................3400 W Corrente: .....................................................................................16 A Tensione: .............................................................................220-240 V Frequenza: ...........................................................................60; 50 Hz Tipo di spina: ..........................................................................Schuko Accessori inclusi: .......1 x Griglia combinata, 1 x Leccarda universale smaltataSerie 6, Forno da incasso, 60 x 60cm, AcciaioHBA257BS0Forno da incasso di moderno ed elegante design con programmi automatici di cottura: per preparare piatti perfetti.- Eco Clean: soffitto, parete posteriore, pareti laterale- Programma di pulizia EcoClean- Display digitale LCD a colore bianco- Programmi automatici: 10- Orologio elettronico con impostazione inizio e fine cottura- Raggiungimento temperatura- Illuminazione interna alogena- Volume cavità: 71 l- <8088brandlookup_nl(TUE,- KIN, SIK, SIB, REW, STA, TKS)>- Ventola tangenziale di raffreddamento- Assorbimento massimo elettrico: 3.4 kW- Dimensioni apparecchio (AxLxP): 595 mm x 594 mm x 548 mm- Dimensioni nicchia (AxLxP): 560 mm - 568 mm x 585 mm - 595 mm x 550 mm- Si prega di fare riferimento alle quote d'installazione mostrate nel disegno tecnicoEtichetta energetica- Classe di efficienza energetica (acc. EU Nr. 65/2014): A(in una scala di classi di efficienza energetica da A+++ a D)- Consumo energetico per ciclo durante funzionamento convenzionale:0.97 kWh- Consumo energetico per ciclo durante funzionamento ventilato:0.81 kWh- Numero di cavità: 1 Tipo di alimentazione: elettrica Volume della cavità:71 lDimensioniSerie 6, Forno da incasso, 60 x 60cm, Acciaio HBA257BS0。
LentiPac HIV表达包装试剂盒-使用手册-130902
Lenti-Pac TM HIV表达包装试剂盒——优化重组慢病毒·使用手册目录1 产品概述2 试剂盒组成3 其它所需细胞及试剂4 实验前准备5 制备慢病毒颗粒6 病毒颗粒滴度检验7 以制得慢病毒颗粒感染目的细胞8 使用许可与质量保证GeneCopoeia, Inc. 广州复能基因有限公司广州高新技术产业开发区广州科学城掬泉路3号广州国际企业孵化器D区8楼邮编：510663电话：4006-020-200邮箱：sales@网址：© 2013 GeneCopoeia, Inc.1.产品概述复能基因现提供40000多套人源及小鼠源ORF表达克隆，同时提供针对人源、小鼠源、大鼠源及其他哺乳类动物染色体组靶标基因的小发夹结构RNAi（shRNA）克隆。
HIV（人类免疫缺陷病毒）载体是近来使用最普遍的慢病毒表达载体，它能有效从体外或体内介导外源基因转导进入多种分化或非分化哺乳动物细胞。
慢病毒表达载体可整合到靶细胞基因组，产生稳定的转基因表达。
根据疾病控制中心作出的标准，复能基因第三代HIV慢病毒载体需要生物安全第二级的实验室操作。
复能基因提供的Lenti-Pac TM HIV表达包装系统包括：优化的慢病毒包装质粒混合物、eGFP阳性对照质粒、可优化病毒产物的复能基因EndoFectin TM转染试剂、可5-10倍提高病毒滴度的复能基因TiterBoost TM滴度增强剂。
结合复能基因HIV载体的慢病毒克隆，制得慢病毒颗粒具有高滴度及高表达水平的特质。
Lenti-Pac TM HIV骨架的表达包装系统可保证重组转录本在哺乳动物细胞的表达。
OmicsLink TM慢病毒ORF表达克隆和shRNA克隆的优点·从体外或体内高效转导基因至所有类型的哺乳动物细胞·ORF表达克隆转导后获得高表达水平·高效敲除靶标mRNA转录本·病毒载体自身失活，不产生其它病毒复制2．试剂盒组成试剂盒组成及推荐储存环境(表格内容参照货号Cat. No. HPK-LvTR-20/HPK-LvTR-40)3. 其它所需细胞及试剂1. GeneCopoeia GCI-L3化学感受态细胞(GeneCopoeia Cat No. STK300-10)，亦可选择Stbl3™化学感受态细胞(Invitrogen Cat No. C7373-03)2. GeneCopoeia 293Ta 慢病毒包装细胞系(GeneCopoeia Cat No. Clv-PK-01)，亦可选择HEK293T/17细胞系(ATCC Cat No. CRL-11268)3. H1299细胞系(ATCC Cat No. CRL-5803)或HT-1080细胞系(ATCC Cat No. CCL-121)，该细胞系用于测定慢病毒滴度。
ViralSEQ Lentivirus Physical Titer Kit说明书
ViralSEQ™ Lentivirus Physical Titer KitCatalog Numbers A52597 and A52598Pub. No. MAN0026127 Rev. A.0Note: For safety and biohazard guidelines, see the “Safety” appendix in the ViralSEQ™ Lentivirus Titer Kits User Guide(Pub. No. MAN0026126). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.Product descriptionThe Applied Biosystems™ ViralSEQ™ Lentivirus Physical Titer Kit is a TaqMan™-based RT-qPCR kit. The kit measures viral count based on highly sensitive viral RNA quantitation from the supernatants of cell-based, bioproduction systems. Viral titers of 104 to 1011 viral particles (VP) per mL can be quantitated using a standard curve generated from the synthetic RNA control included with the kit. Lentivirus quantitation by RT-qPCR is accurate, sensitive, and reproducible.The ViralSEQ™ Lentivirus Physical Titer Kit is compatible with the PrepSEQ™ Nucleic Acid Sample Preparation Kit (Cat. A50485), which offers both a manual and automated sample preparation workflow. For real‑time PCR, the ViralSEQ™ Lentivirus Physical Titer Kit has been validated on the Applied Biosystems™ 7500 Fast Real-Time PCR System and the Applied Biosystems™ QuantStudio™ 5 Real‑Time PCR System. Data analysis is streamlined using AccuSEQ™ Real‑Time PCR Software that provides accurate quantitation and security, audit, and e-signature capabilities to help enable 21 CFR Pt 11 compliance.For more information about reagent use, see the ViralSEQ™ Lentivirus Titer Kits User Guide (Pub. No. MAN0026126).Treat samples with DNase I, RNase‑free (1 U/µL)DNase I, RNase‑free (1 U/µL) treatment is used to digest double-stranded DNA.Thaw all reagents on ice. Invert the DNase I, RNase‑free (1 U/µL) several times to mix, then centrifuge briefly. All other reagents should be vortexed, then centrifuged briefly before use.1.Set up the DNase I, RNase‑free (1 U/µL) reactions in a MicroAmp™ Optical 96-Well Reaction Plate (0.2 mL).[1]Mix gently by pipetting 3-5 times when adding.2.Mix the reactions by gently pipetting up and down 5 times, then seal the reaction plate with MicroAmp™ Clear Adhesive Film.3.Centrifuge the plate at 1,000 x g for 2 minutes.4.Load the reactions onto the VeritiPro™ 96-well Thermal Cycler, then start the DNase I treatment.Set cover temperature: 105℃Set reaction volume: 18 µL[1]Do not hold for more than 5 minutes. Proceed immediately to DNase I inactivation.5.Centrifuge the plate at 1,000 x g for 2 minutes.CAUTION! The plate is in contact with the heated lid. Remove carefully.6.Gently remove the MicroAmp™ Clear Adhesive Film, then discard.IMPORTANT! Do not touch wells when removing the MicroAmp™ Clear Adhesive Film. Contamination can lead to inaccurate results.7.Add 2 µL of 50mM EDTA to each reaction well. Mix by gently pipetting 5 times with a P10/P20 pipettor set to 10 µL.8.Seal the reaction plate with MicroAmp™ Clear Adhesive Film, then centrifuge the plate at 1,000 x g for 2 minutes.9.Load the reactions onto the VeritiPro™ 96-well Thermal Cycler, then start the DNase I inactivation.Set cover temperature: 105℃Set reaction volume: 20 µL[1]Do not hold for more than 5 minutes.10.Centrifuge the plate at 1,000 x g for 2 minutes.CAUTION! The plate is in contact with the heated lid. Remove carefully.IMPORTANT! Do not vortex.Place the plate on ice until use.Prepare the serial dilutionsThaw the Physical Titer RNA Control (2 ✕1010 copies/µL) on ice. Vortex at medium speed for 5 seconds, briefly centrifuge, then place on ice until use.bel nonstick 1.5‑mL microfuge tubes: NTC, SD1, SD2, SD3, SD4, SD5, and SD6 [used for limit of detection (LOD)].2.Add 35 µL of RNA Dilution Buffer (RDB) to the NTC (no template control) tube. Place the tube on ice.3.Perform the serial dilutions.When dispensing RNA, pipette up and down gently. After each transfer, vortex for 7 seconds, then centrifuge briefly.Table 1 Standard curve dilutions (ViralSEQ™ Lentivirus Physical Titer Kit)Store the standard curve dilution tubes at 4°C or on ice. Use the dilutions within 6 hours for RT-qPCR.Prepare the kit reagents and premix solutionThaw all kit reagents on ice. Vortex the reagents for 5 seconds, briefly centrifuge, then place the reagents on ice until use.bel a microcentrifuge tube for the Premix Solution.2.Prepare the Premix Solution according to the following tables.IMPORTANT! Use a separate pipette tip for each component.Table 2 Premix Solution[1]Includes 10% excess to compensate for pipetting loss.3.Vortex the Premix Solution for 10 seconds to mix, then briefly centrifuge.Store the Premix Solution at 4°C or on ice until use.Prepare the PCR reactionsPlace the plate containing DNase I-treated samples on a MicroAmp™ 96-Well Base, then gently remove the MicroAmp™ Clear Adhesive Film. Gently pipette up and down 3 times to mix the samples.1.Dispense the following into the appropriate wells of a MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 mL, gently pipetting at thebottom of the well.Figure 1 Recommended plate layout2.Seal the plate with MicroAmp ™Optical Adhesive Film.3.Vortex the reaction plate for 10 seconds, then centrifuge at 1,000 x g for 2 minutes.Note: Ensure there are no bubbles in the reaction wells. If present, tap the well gently to remove bubbles, then re-centrifuge.Proceed immediately to “Start the run (QuantStudio ™ 5 Real ‑Time PCR Instrument)”.Create a ViralSEQ ™ templateCreate a new template in the (Home) screen of the AccuSEQ ™Real ‑Time PCR Software v3.1.1.ClickCreate New on the home screen.Create New ExperimentpaneFactory default/Admin Defined Templates —List of existing default or Admin Defined templates. These templates can beused as templates for new experiments.My Templates —List of templates available to the user that is signed in. These templates can be used as templates for newexperiments.Create New —Used to create an experiment or template with nopre-existing settings.Create My Template —Used to create a new template (stored locally in My Templates).Create Admin Defined Template —Used to create a new template (Administrator only).2.Select Create My Template or Create Admin Defined Template .3.Edit the Experiment Properties as required.a.In the Template Name field, modify the template name. For example, LV Titer template.b.(Optional ) Enter information in the Comments field.c.In the Setup tab, select:•Experiment Type —Quantitation-Standard Curve •Chemistry —TaqMan ® Reagents•Ramp Speed —Standard-2hrs •Block Type —96-Well 0.1mL Blockd.(Optional ) Select Is Locked to lock the template. If locked, users are unable to edit the template.4.ClickAnalysis settings to change the default C t Settings and Flag Settings.a.In the C t Settings tab, click Edit Default Settings .b.Deselect Automatic Threshold , then enter 0.200.c.Ensure that Automatic Baseline is selected.d.Click Save Changes .e.Deselect Default Settings , then click Applyto save any changes before closing the window.2C tSettingsFlag SettingsEdit Default SettingsbuttonDefault Settingscheckbox Apply buttonf.In the Flag Settings tab, deselect the following flags.•CQCONF —Low Cq confidence •EXPFAIL —Exponential algorithm failed •NOAMP —No amplification •NOSIGNAL —No signal in wellNote: Use the scrollbar on the right to scroll down the list of flags.g.Click Apply to save any changes before closing the window.5.Click Next .Template name cannot be changed after this step.The qPCR Method screen is displayed.Edit the run method and optical filter selectionThis section provides general procedures to edit the run method and optical filter selection in the qPCR Method. To edit the default run method, see the AccuSEQ™ Real‑Time PCR Software v3.1 User Guide (Pub. No. 100094287).1.Set the reaction volume to 25 µL.2.Edit Step 1 of the Hold Stage to 45℃ for 30 minutes.3.Set Step 2 of the Hold Stage to 95℃ for 10 minutes.4.Set Step 1 of the PCR Stage to 95℃ for 15 seconds.5.Edit Step 2 of the PCR Stage to 60℃ for 45 seconds.6.Set the cycle number to 40.7.Ensure that Data Collection occurs after Step 2.123Figure 2 Lentivirus Physical Titer Run MethodReaction volume- set to 25µLStageCycle number- set to 40 cycles8.(Optional) Click (Optical Filter Settings) to view the default filter settings.•The default optical filter selection is suitable for the ViralSEQ™ Lentivirus Physical Titer Kit.•The ViralSEQ™ Lentivirus Physical Titer Kit requires the QuantStudio™ 5 System to be calibrated for FAM™, VIC™, and ROX™.•For more information about system dyes and their calibration and optical filter selection, see QuantStudio™ 3 and 5 Real‑Time PCR Systems Installation, Use, and Maintenance Guide (Pub. No. MAN0010407).9.Click Next.Assign plate and well attributesNote: This section provides general procedures to set up the plate.For specific instructions for each assay type, see the corresponding chapter in this guide. Do not change Targets for default assay templates.TargetsSamplesPlate setup toolbarSelect Item to highlight (Sample, Target, or Task).Select Item. For example, Sample 1. Sample 1 replicates arehighlighted.Define& setup Standard(View Legend)(Print Preview)View (Grid View or Table View)1.In Plate Setup screen, click or click‑drag to select plate wells in the (Grid View) of the plate.2.Assign the well attributes for the selected wells. Each well should have a Sample Name under Samples, as well as the appropriateTargets under Targets. Reporters should be FAM™ dye for Lentivirus Physical Titer, and VIC™ dye for internal positive control (IPC).a.To add new Samples or Targets, click Add in the appropriate column on the left of the screen, then edit the new Name andother properties as required. The new sample or target is then selectable within the wells of the plate.b.For each sample (e.g. DNase-treated lentivirus sample, standard curve dilution, or NTC), two targets should be included.•Select the FAM™ dye reporter for Lentivirus Physical Titer detection.•Select the VIC™ dye for IPC detection.c.Select NFQ-MGB as the quencher for both targets.d.For standard curve dilution samples (SD1 to SD5), the Task for Lentivirus Physical Titer target should be indicated as “S” forStandard, with the appropriate copy number written under Quantity. For instance, the quantity for SD1 is 1E9 copies. Change the Task by clicking on the field and using the drop-down menu. Copy numbers can be indicated using scientific notation (e.g.“1E9”) and the program will convert it to numerical format.e.For DNase-treated samples and SD6, set the Task for Lentivirus Physical Titer target to U for Unknown.f.For NTC wells, set the Task for Lentivirus Physical Titer target to N for NTC.g.For IPC wells, set the Task for Lentivirus Physical Titer target to U for Unknown.h.To change sample names, click the name in the Name column, then type the new name. To change Reporters and Quenchers,click the dye, then select from the dropdown list.When a Sample or Targetname are edited, two entries are added to the Audit trail (one for Delete, and another for Create).23AddbuttonCheckbox—Select Targets and Samples to go in the selectedwell.Textbox—Click the name to edit.Scrollbar—Use to scroll to additional properties.•Use the plate setup toolbar (above the plate) to make edits to the plate.–Click View to show/hide the Sample Name, Sample Color, or Target from the view.•To add consecutive samples (with the same Target ), select a well, then click ‑drag the dark blue box to the right.3.(Optional ) Double ‑click a well to enter comments for the selected well.4.Select ROX ™dye from the Passive Reference drop-down list (bottom left of screen).5.Click Save to save the template.This template can then be used to create experiments.Start the run (QuantStudio ™ 5 Real ‑Time PCR Instrument)Ensure that the plate is loaded in the QuantStudio ™5 Real ‑Time PCR Instrument.™A message stating Run has been started successfully is displayed when the run has started.Review the resultsAfter the qPCR run is finished, use the following general procedure to analyze the results. For more detailed instructions see theAccuSEQ ™Real ‑Time PCR Software v3.1 User Guide (Pub. No. 100094287).1.In the AccuSEQ ™Real ‑Time PCR Software, open your experiment, then navigate to the Result tab.ResulttabAnalysis SettingsPlot horizontal scrollbar Analyze button2.In the Result Analysis tab, select individual targets, then review the Amplification Curve plots for amplification profiles in thecontrols, samples, and the standard curve. Ensure that threshold is set to 0.200 with an automatic baseline.3.In the Result Analysis tab, review the QC Summary for any flags in wells.4.In the Result Analysis tab, review the Standard Curve plot. Verify the values for the Slope, Y ‑intercept, R 2, and Efficiency are withinacceptable limits.Note: The Standard Curve efficiency should be between 90-110% and the R 2>0.99. If these criteria are not met, up to two points,not in the same triplicate, can be removed from the standard curve data, and the analysis repeated.5.In Table View, ensure that C t values are within the standard curve range.•Samples with C t values that exceed the upper limit of quantitation (109 copies) of the standard curve should be diluted and re-run.•Samples with C t values that exceed the lower limit of detection (LoD of 10 copies) and IPC shows no signs of PCR inhibition,suggests the absence of lentivirus.6.(Optional ) Outliers can be excluded from the results. To exclude, select the well, then click Omit/Include , then reanalyze by clickingAnalyze .7.(Optional ) Select File 4Print Report to generate a hard copy of the experiment, or click Print Preview to view and save the report asa PDF or HTML file.8.Export the results.a.Navigate to the Report tab.b.Check all boxes under Contents .c.Select Export Data in One File .d.Select the XLS format, then click Export .Calculate the titer (VP/mL)1.Download the Physical Titer Calculation Tool .a.Go to .b.Search for the ViralSEQ ™Lentivirus Physical Titer Kit.c.Download the tool from the Documents section.2.Open the tool, then follow the instructions in the tool to calculate the titer.Calculate lentivirus titers from qPCR dataTo determine the number of lentivirus RNA copies per mL in the original sample, the copy numbers obtained from the qPCR must be multiplied by the dilution factor of the sample during extraction and DNase I treatment. Since there are 2 copies of RNA/target per lentivirus particle, the number of viral particles per mL (VP/mL) is 0.5x the number of lentivirus RNA copies.Viral particles per mL =qPCR copies x sample dilution factor x 0.5Volume of sample used (mL)For help in determining the qPCR copy numbers, see the QuantStudio ™Design and Analysis Desktop Software User Guide (Pub. No. MAN0010408).For example, if the following parameters were used,•10 µL of lentivirus culture was extracted with the KingFisher ™Flex Purification System with 96 Deep-Well Head and eluted in 200 µL •10 µL of this eluate (20x dilution) was treated with DNase I, RNase ‑free (1 U/µL) in a total volume of 20 µL.• 5 µL of the DNase-treated sample (4x dilution) was used for the qPCR reaction.Lentivirus sample10 μL Sample Extraction Elute: 200 μL DNaseI Treat Total: 20 μL reaction RT-qPCRTotal: 25 μL reactionUse 10 μL (1:20 dilution)Use 5 μL (1:4 dilution)then, the calculation would be:Viral particles per mL =qPCR copies x (20x4) x 0.50.01 (mL)Note: qPCR can only determine the number of physical particles in a virus culture. To determine the numbers of infectious units,cell-based transduction experiments must be carried out. The titers of physical particles are often higher than infectious titers by 10-1000fold, depending on the purity of the lentivirus preparation and the levels of infectious particles within the culture.Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at /us/en/home/global/terms-and-conditions.html . If you have any questions, please contact Life Technologies at /support .Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United KingdomThe information in this guide is subject to change without notice.DISCLAIMER : TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information : These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Vortex-Genie is a trademark of Scientific Industries. Windows and Excel are trademarks of Microsoft Corporation. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license./support | /askaquestion 23 August 2022。
MKP型号电容器产品说明书
Dimensions: [mm]Scale - 1:2MDBP525256JA20DCPP15040890744429002CSMDBP525256JA20DCPP15040 890744429002CSMDBP525256JA20DCPP15040 890744429002CSMDBP525256JA20DCPP15040 890744429002CST e m p e r a t u r eT T T MDBP525256JA20DCPP15040890744429002CSCautions and Warnings:The following conditions apply to all goods within the product series of Film Capacitors of Würth Elektronik eiSos GmbH & Co. KG:General:•This electronic component is designed and manufactured for use in general electronic equipment.•Würth Elektronik must be asked for a written approval (following the certain PPAP level procedure) before incorporating the components into any equipment in the field such as military, aerospace, aviation, nuclear control, submarine, transportation (automotive control, train control, ship control), transportation signal, disaster prevention, medical, public information network etc. where higher safety and reliability are especially required and/or if there is the possibility of direct damage or human injury.•Electronic components that will be used in safety-critical or high-reliability applications, should be pre-evaluated by the customer. •Direct mechanical impact to the product shall be prevented as material of the body, pins or termination could flake or in the worst case it could break.•Avoid any water or heavy dust on capacitors surface, which may cause electrical leakage, damage, overheating or corrosion.•Würth Elektronik products are qualified according to international standards, which are listed in each product reliability report. Würth Elektronik does not warrant any customer qualified product characteristic, beyond Würth Elektronik specifications, for its validity and sustainability over time.•The customer is responsible for the functionality of his or her own products. All technical specifications for standard products also apply to customer specific products.•The component is designed and manufactured to be used within the datasheet specified values. If the usage and operation conditions specified in the datasheet are not met, the body, pins or termination may be damaged or dissolved.•Do not apply any kind of flexural or compressive force onto soldered or unsoldered component.•The capacitance tolerance as specified within the datasheet is only valid on the date of delivery and according specified measurement criteria.Product specificStorage conditions• A storage of Würth Elektronik products for longer than 12 months is not recommended. Within other effects, the terminals may suffer degradation, resulting in bad solderability. Therefore, all products shall be used within the period of 12 months based on the day of shipment.•Do not expose the components into direct sunlight.•The storage condition in the original packaging is defined according to DIN EN 61760-2.•The environment in which the capacitors are operated and stored has to have atmospheric characteristics and must be free of dew condensation and toxic gases (e.g. chlorine, ammonia, sulfur, hydrogen sulphide and hydrogen sulfate).•Do not expose the capacitor to environments with hazardous gas, ozone, ultraviolet rays or any kind of radiation. Avoid any contact of the capacitor with direct sunshine, saltwater, spray of water or types of oil during storage. •The storage conditions stated in the original packaging apply to the storage time and not to the transportation time of the components. Operating climatic conditions•Do not exceed the lower nor the upper specified temperature under no circumstances.•Do not use the capacitors under high humidity, high temperature or under high or low atmospheric pressure which may affect capacitors reliability.•Surface temperature including self-heating must be kept below the maximum operating temperature.Operating load conditions•Due to self-heating the reliability of the capacitor may be reduced, if high frequency AC or pulse is applied.•Consider carefully possible specific changes of electrical characteristics like capacitance over temperature, voltage and time as well as the specific performance over frequency for the actual use conditions.•Avoid any overvoltage and do not apply a continuous overvoltage. If an overvoltage is applied to the capacitor, the leakage current can increase drastically. The applied working voltage is not allowed to exceed the rated working voltage of the specific capacitor.•If film capacitors with safety approvals are operated with a DC voltage exceeding the specified AC voltage, the approvals given on the basis of IEC 60384-14 are no longer valid.•For the WCAP-FTDB film capacitor the maximum peak voltage Vpeak+ shall not be greater than the rated voltage VR according to the temperature derating of the rated voltage VR. The peak-to-peak value of the ripple voltage Vp-p should not be greater than 0.3*VR according to the temperature derating of the rated voltage VR. The rated voltage of the capacitor may need to be reduced for different operating temperatures. See voltage derating curve within this datasheet.Packaging:•The packaging specifications apply only to purchase orders comprising whole packaging units. If the ordered quantity exceeds or is lower than the specified packaging unit, packaging in accordance with the packaging specifications cannot be ensured. Soldering•The solder profile must comply with the Würth Elektronik technical soldering specification. All other profiles will void the warranty. •All other soldering methods are at the customer’s own risk.•Strong forces which may affect the coplanarity of the component’s electrical connection with the PCB (i.e. pins), can damage the part, resulting in void of the warranty.•Customer needs to ensure that the applied solder paste, the paste thickness and solder conditions are enough to guarantee a sufficient solder result according to the relevant criteria of IPC-A-610.•Excessive amount of solder may lead to higher tensile force and chip cracking. Insufficient amount of solder may detach the capacitor due to defective contacts.•Do not use excessive nor insufficient flux.Würth Elektronik eiSos GmbH & Co. KGEMC & Inductive SolutionsMax-Eyth-Str. 174638 WaldenburgGermanyCHECKED REVISION DATE (YYYY-MM-DD)GENERAL TOLERANCE PROJECTIONMETHODFPu001.0002022-10-13DIN ISO 2768-1mDESCRIPTION TECHNICAL REFERENCEWCAP-FTDB DC-Link Capacitor MDBP525256JA20DCPP15040ORDER CODE890744429002CSSIZE/TYPE BUSINESS UNIT STATUS PAGECleaning•Do not use any other cleaning solvents for box-typed capacitors except: ethanol, isopropanol, n-propanol - water mixtures. After cleaning a drying process with temperatures not exceeding 65°C and not longer than 4 hours is mandatory to prevent any kind of electrical damage.Coating, molding and potting of the PCB•If the product is potted in the costumer’s application, the potting material might shrink or expand during and after hardening. Shrinking could lead to an incomplete seal, allowing contaminants into the body and termination. Expansion could damage the body or termination. We recommend a manual inspection after potting to avoid these effects.•If final assemblies will be placed completely in any plastic resin, physical, chemical and thermal influences must be considered. •When coating and molding the PCB, verify the quality influence on the capacitor.•Verify the curing temperature and assure that there is no harmful decomposing or reaction gas emission during curing. •Do not exceed the specified max. self-heating.Vibration resistance•Do not exceed the vibration limits given by IEC60068-2-6.Handling•After soldering, please pay attention not to bend, twist or distort the PCB in handling and storage. •Avoid excessive pressure during the functional check of the PCB. •Avoid bending stress while breaking the PCB.•WCAP-FTXX and WCAP-FTX2 capacitors are not designed and not recommended to be used in series connection to the mains. •The temperature rise of the component must be taken into consideration. The operating temperature is comprised of ambient temperature and temperature rise of the component.The operating temperature of the component shall not exceed the maximum temperature specified.Flammability•Avoid any external energy or open fire (passive flammability).These cautions and warnings comply with the state of the scientific and technical knowledge and are believed to be accurate and reliable.However, no responsibility is assumed for inaccuracies or incompleteness.(V2.2)Würth Elektronik eiSos GmbH & Co. KG EMC & Inductive Solutions Max-Eyth-Str. 174638 Waldenburg GermanyCHECKED REVISION DATE (YYYY-MM-DD)GENERAL TOLERANCEPROJECTION METHODFPu001.0002022-10-13DIN ISO 2768-1mDESCRIPTIONTECHNICAL REFERENCEWCAP-FTDB DC-Link CapacitorMDBP525256JA20DCPP15040ORDER CODE890744429002CSSIZE/TYPEBUSINESS UNITSTATUSPAGEImportant NotesThe following conditions apply to all goods within the product range of Würth Elektronik eiSos GmbH & Co. KG:1. General Customer ResponsibilitySome goods within the product range of Würth Elektronik eiSos GmbH & Co. KG contain statements regarding general suitability for certain application areas. These statements about suitability are based on our knowledge and experience of typical requirements concerning the areas, serve as general guidance and cannot be estimated as binding statements about the suitability for a customer application. The responsibility for the applicability and use in a particular customer design is always solely within the authority of the customer. Due to this fact it is up to the customer to evaluate, where appropriate to investigate and decide whether the device with the specific product characteristics described in the product specification is valid and suitable for the respective customer application or not.2. Customer Responsibility related to Specific, in particular Safety-Relevant ApplicationsIt has to be clearly pointed out that the possibility of a malfunction of electronic components or failure before the end of the usual lifetime cannot be completely eliminated in the current state of the art, even if the products are operated within the range of the specifications.In certain customer applications requiring a very high level of safety and especially in customer applications in which the malfunction or failure of an electronic component could endanger human life or health it must be ensured by most advanced technological aid of suitable design of the customer application that no injury or damage is caused to third parties in the event of malfunction or failure of an electronic component. Therefore, customer is cautioned to verify that data sheets are current before placing orders. The current data sheets can be downloaded at .3. Best Care and AttentionAny product-specific notes, cautions and warnings must be strictly observed. Any disregard will result in the loss of warranty.4. Customer Support for Product SpecificationsSome products within the product range may contain substances which are subject to restrictions in certain jurisdictions in order to serve specific technical requirements. Necessary information is available on request. In this case the field sales engineer or the internal sales person in charge should be contacted who will be happy to support in this matter.5. Product R&DDue to constant product improvement product specifications may change from time to time. As a standard reporting procedure of the Product Change Notification (PCN) according to the JEDEC-Standard inform about minor and major changes. In case of further queries regarding the PCN, the field sales engineer or the internal sales person in charge should be contacted. The basic responsibility of the customer as per Section 1 and 2 remains unaffected.6. Product Life CycleDue to technical progress and economical evaluation we also reserve the right to discontinue production and delivery of products. As a standard reporting procedure of the Product Termination Notification (PTN) according to the JEDEC-Standard we will inform at an early stage about inevitable product discontinuance. According to this we cannot guarantee that all products within our product range will always be available. Therefore it needs to be verified with the field sales engineer or the internal sales person in charge about the current product availability expectancy before or when the product for application design-in disposal is considered. The approach named above does not apply in the case of individual agreements deviating from the foregoing for customer-specific products.7. Property RightsAll the rights for contractual products produced by Würth Elektronik eiSos GmbH & Co. KG on the basis of ideas, development contracts as well as models or templates that are subject to copyright, patent or commercial protection supplied to the customer will remain with Würth Elektronik eiSos GmbH & Co. KG. Würth Elektronik eiSos GmbH & Co. KG does not warrant or represent that any license, either expressed or implied, is granted under any patent right, copyright, mask work right, or other intellectual property right relating to any combination, application, or process in which Würth Elektronik eiSos GmbH & Co. KG components or services are used.8. General Terms and ConditionsUnless otherwise agreed in individual contracts, all orders are subject to the current version of the “General Terms and Conditions of Würth Elektronik eiSos Group”, last version available at .Würth Elektronik eiSos GmbH & Co. KGEMC & Inductive SolutionsMax-Eyth-Str. 174638 WaldenburgGermanyCHECKED REVISION DATE (YYYY-MM-DD)GENERAL TOLERANCE PROJECTIONMETHODFPu001.0002022-10-13DIN ISO 2768-1mDESCRIPTION TECHNICAL REFERENCEWCAP-FTDB DC-Link Capacitor MDBP525256JA20DCPP15040ORDER CODE890744429002CSSIZE/TYPE BUSINESS UNIT STATUS PAGE。
常规真核表达与鉴定
4、再以1mL PBS洗涤离心细胞沉淀，在管口沿壁加入，吹起 沉淀；离心：4℃、12000r/m、2min；弃上清。
5、每个离心管加入100uL细胞裂解液(RIPA)，吹打混匀，冰 上作用30min裂解细胞。
6、离心4℃ 12000r/m、5min；吸取上清约30uL；煮样，加 上样loading buffer，蛋白电泳。
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二、常用真核表达系统
1、毕赤酵母表达体系：酵母细胞表达系统蛋白表达水平高， 生产成本低，但纯化难度大。
2、慢病毒表达系统：商品化的慢病毒表达系统（如Lenti-X） 在病毒包装时获得VSV-G泛嗜性重组慢病毒，通过膜质偶联 和膜质融合的方式感染靶细胞，可感染几乎所有类型的哺 乳动物细胞。
3、哺乳动物细胞表达体系：对蛋白的加工与修饰与酵母及昆 虫表达系统完全不同，可通过脂质体等直接将外源表达质 粒导入哺乳动物细胞，进行表达。哺乳动物细胞产生的蛋 白质更接近于天然蛋白，但其表达量低。
3、某些特殊的表达载体：如反向遗传表达载体，昆虫表达 系统载体等。
5
四、哺乳动物细胞转染
1、引物设计：构建重组真核表达质粒，一般在引物设计时 首先会在引物中引物一些能够促进蛋白表达（如Kozak序 列）或者便于后期检测的序列或标签（如flag、HA、His 等），注意这些序列在引物中的位置。
2、实验材料：真核表达细胞系、DMEM、FBS、OPTI-MEM、 PBS、trypsin、携带有外源基因的重组质粒（去细胞内毒 素法抽提）、转染试剂（或仪器）、细胞计数器、盖玻片、 六孔板等。
3、转染细胞密度约2～5×106/mL,一般在细胞铺板后12～18h 内进行转染，转染的质粒要测定浓度，按2ug/孔进行。
6
细胞转染步骤
慢病毒包装、纯化、滴度测定及感染
慢病毒包装、纯化、滴度测定及感染一、包装1.包装细胞Lenti-X?LentiviralExpressionSystemsUserManual.pdf（P11）;Lenti-X?shRNAExpressionSystemsUserManual.pdf(P16)2.病毒载体及包装质粒病毒载体：DNA组构建及中量提取；包装质粒：商品化产品（Lenti-X?LentiviralExpressionSystemsUserManual.pdf （P12）;Lenti-X?shRNAExpressionSystemsUserManual.pdf(P17)）或DNA中量提取（目前唯一使用来源）；3.细胞转染方法一：Lenti-X?LentiviralExpressionSystemsUserManual.pdf（P12）;Lenti-X?shRNAExpressionSystemsUserManual.pdf(P17)；方法二：按照Lipofectamine?LTX&PlusReagent进行，简要中文说明如下：a.转染前24小时，把4–5x106个293T细胞传代至10cm培养皿中，加入完全培养基至终体积10ml，培养过夜，转染时，细胞密度为80–90%；b.293T细胞转染前2小时换上9ml新鲜的完全培养基；c.用之前充分混匀PLUSReagent，在EP管中加入15ug的DNA混合物(pLVX-shRNAPlasmidDNA：pMDLg：pRes-Rev：pCMV-VSV-G的摩尔比为1：1：1：1)，15ul的PLUSReagent，用Opti-MEM定容到500ul，标记为Tube1，温和混匀，室温孵育5分钟；d.充分混匀MixLipofectamineLTX，在EP管中加入45ul的MixLipofectamineLTX和455ul的Opti-MEM，标记为Tube2，温和混匀；e.将Tube1的溶液加到Tube2中，温和混匀，室温孵育5分钟；Tube1(PlasmidDNA) Tube2(LTX)15ugDNAMIX(pLVX-shRNAPlasmidDNA：455μl Opti-MEMpMDLg：pRes-Rev：pCMV-VSV-G=1:1:1:1)15μl PLUSReagent 45μl MixLipofectamineLTX Upto500μl Opti-MEM 500μlTotalVolume500μlTotalVolumef.将1ml转染复合物逐滴加入前一天种好细胞的100mm皿中，边加边摇匀。
Lenti-Pac HIV 慢病毒包装试剂盒 使用手册说明书
Lenti-Pac™HIV慢病毒包装试剂盒使用手册目录1产品概述2试剂盒组成3包装慢病毒颗粒所需的实验材料4慢病毒颗粒转导靶细胞所需的实验材料5实验前的准备6慢病毒颗粒的包装制备原理7制备慢病毒8慢病毒滴度检验9慢病毒感染目的细胞10使用许可与质量保证GeneCopoeia,Inc.广州易锦生物技术有限公司广州高新技术产业开发区广州科学城掬泉路3号广州国际企业孵化器F区8楼邮编：510663电话：4006-020-200邮箱：******************英文网址：中文网址：©2022GeneCopoeia,Inc.HIV（人类免疫缺陷病毒）骨架的慢病毒载体是目前最常用的慢病毒表达载体，它能在体外或体内实验有效介导外源基因转导多种分化或非分化哺乳动物细胞，使外源基因稳定整合靶细胞的基因组，并进行稳定的高水平表达。
GeneCopoeia Lenti-Pac™HIV慢病毒包装系统是安全性较高的第三代慢病毒包装系统，表达载体自身失活，不产生额外的慢病毒复制。
该系统包括：混合包装质粒、EndoFectin™-Lenti转染试剂、可进一步提高滴度的TiterBoost™滴度增强剂、以及eGFP阳性对照质粒。
Lenti-Pac™HIV慢病毒包装系统可将HIV载体的慢病毒表达克隆包装成为具有转导效果的慢病毒颗粒，包装所得的慢病毒颗粒可立即使用，介导目的基因在哺乳动物细胞进行高效表达。
GeneCopoeia提供40000多种人源及小鼠源的慢病毒载体ORF表达克隆，以及针对人源、小鼠源、大鼠源及其他哺乳类动物染色体组靶基因的慢病毒载体shRNA克隆。
除此以外，GeneCopoeia同时提供20000多种人源慢病毒载体miRNA前体表达克隆、miRNA抑制剂表达克隆以及启动子报告克隆，18000种小鼠的慢病毒载体启动子报告克隆。
以上所有克隆均应用HIV骨架，可随时应用于慢病毒包装。
根据疾病控制中心制定的标准，GeneCopoeia第三代HIV慢病毒包装系统满足生物安全第二级别（BSL-2）的要求。
Lavastoviglie Serie 4 60 cm Inox SGI4HCS48E 商品说明书
Serie 4, Lavastoviglie integrabile, 60 cm, acciaio inoxSGI4HCS48EAccessori specialiSGZ0IC00 :SGZ1010 Prolunga acqua-stop sgs/gi/gv/guSMZ1051EU :SMZ2044 Frontale dello sportello e zoccolo niroSMZ5003 Cerniera ribaltabile per dimensioni alte SMZ5005 Kit di adattamento e fissaggio NiroSMZ5035 :SMZ5100 Cestello porta posateSMZ5300 Accessorio lavastoviglie da incasso Carica facilmente le tue stoviglie conl'opzione di asciugatura extra al tocco di un pulsante.• ExtraDry: opzione selezionabile per un'ulteriore asciugatura.• CestelloVario - Flessibilità nel terzo livello di carico• Il programma Silenzio: il modo più silenzioso per far funzionarela lavastoviglie.• AquaStop: protezione antiallagamento, con una garanzia a vita al 100%.• EcoSilence Drive: elevato risparmio energetico, silenziosità di funzionamento e vita utile particolarmente lunga.Dati tecniciClasse di efficienza energetica: ........................................................D Consumo energetico del programma Eco per 100 cicli : .........85 kWh Numero massimo di coperti: .. (14)Consumo di acqua del programma eco in litri per ciclo: ..............9.5 l Durata del programma: .............................................................4:55 h Emissioni di rumore aereo: ......................................44 dB(A) re 1 pW Classe di emissioni di rumore aereo: ................................................B Da incasso / a libera installazione: .....................................Da incasso Altezza senza piano di lavoro: ....................................................0 mm Dimensioni (lxp): ................................................815 x 598 x 573 mm Dimensioni del vano per l'installazione: .......815-875 x 600 x 550 mm Profondità con porta aperta a 90 gradi: ...............................1150 mm piedini regolabili: ...................................................Yes - all from front Regolazione massima dei piedini: ............................................60 mm Zoccolo regolabile: ..........................................verticale ed orizontale Peso netto: ..............................................................................35.6 kg Peso lordo: ..............................................................................37.5 kg Potenza: ..................................................................................2400 W Corrente: .....................................................................................10 A Tensione: .............................................................................220-240 V Frequenza: ...........................................................................50; 60 Hz Repair index: ..................................................................................8.1 Lunghezza del cavo di alimentazione elettrica: .....................175.0 cm Tipo di spina: ..........................................................................Schuko Lunghezza tubo entrata: ..........................................................165 cm Lunghezza tubo uscita: ............................................................190 cm Codice EAN: (4242005322183)Installazione: ......................................................................IntegrabileSerie 4, Lavastoviglie integrabile, 60cm, acciaio inoxSGI4HCS48ECarica facilmente le tue stoviglie conl'opzione di asciugatura extra al tocco di un pulsante.Prestazioni e consumo- Classe di efficienza energetica¹: D- Energia² / Acqua³: 85 kWh / 9.5 litri- Capacità: 14 coperti- Durata del programma⁴: 4:55 (h:min)- Livello sonoro: 44 dB(A) re 1 pW- Classe di efficienza di rumore: B- Livello di rumore programma Silence: 41(A) re 1 pWProgrammi e opzioni- 6 Programmi: Eco 50 °C, Auto 45-65°, Intensive 70 °C, Express 65°, Silence- Prelavaggio- 3 opzioni speciali: Extra Dry, Half Load, SpeedPerfectPlus- Programma manutenzioneTecnologia lavaggio- Scambiatore di calore.- DosageAssist- EcoSilence Drive- Automatismo di pulizia- Sistema di filtri autopulenti con ondulazione a 3 livelli- Contenitore interno: Materiale della vasca interna in acciaio inox Sistema Cestelli- Cestelli Flex- VarioCestello- Cestello superiore regolabile in altezza con Rackmatic (3 livelli)- Ruote con scorrimento facile sul cestello inferiore- Cestello inferiore con blocco (rackStopper) per evitare che fuoriesca dalle guide.- Griglie abbattibili nel cestello superiore (2x)- Griglie abbattibili nel cestello inferiore (2x)- Ripiani per tazze nel cestello superiore (2x)- Carrello inferiore con ripiani per tazze (2x)Indicazione e funzionamento- Iscrizioni di testo in chiaro (inglese)- Indicazione tempo residuo (min.)- Programmatore inizio lavaggio (1-24 h)Sicurezza- AquaStop: una garanzia Bosch per danni causati dall'acqua - durata del dispositivo*- Sicurezza bambini (Tasti)- Tecnologia di protezione del vetro- Aiuto per il riempimento del sale (Imbuto)- Protezione contro il vaporeDimensioni - Dimensioni del prodotto (HxLxP): 81.5 x 59.8 x 57.3 cm¹ In una scala di classi di efficienza energetica da A a G² Consumo di energia in kWh per 100 cicli (nel programma Eco 50°C)³ Consumo di acqua in litiri per ciclo (nel programma Eco 50 °C)⁴ Durata del programma Eco 50 °C* Verificare i termini di garanzia al link/ch/it/condizioni-generali-di-garanziaSerie 4, Lavastoviglie integrabile, 60 cm, acciaio inoxSGI4HCS48E。
什么是非整合型慢病毒
什么是非整合型慢病毒常规慢病毒载体能高效感染分裂细胞和非分裂细胞，将外源基因有效地整合到宿主染色体上，从而实现持久性表达。
然而这种随机整合会带来插入突变的风险。
于是，研究人员也期望用到非整合型的慢病毒包装系统。
百恩维生物对整合酶蛋白进行突变，推出了整合酶缺陷型的慢病毒包装系统（非整合型慢病毒）。
非整合型慢病毒在目的细胞中产生了环状附加体，这些载体在分裂细胞中瞬时表达，但在非分裂细胞中稳定表达。
与整合型慢病毒相比，非整合型慢病毒导致插入突变的风险大大降低，适合iPS研究、RNA干扰、同源重组等领域。
非整合型慢病毒包装系统属于第四代的包装系统，它将pol基因也分离出来，使gag、pol和env成为三个独立载体，而不是两个载体，从而大大降低了产生具有复制能力的病毒的可能性，进一步提高了生物安全性。
此系统与其他慢病毒载体配套使用可以制备出高滴度的慢病毒颗粒，无需浓缩即可达到 5x108 IU/ml。
之所以能制备高滴度的病毒颗粒，主要有以下三个关键因素。
首先，非整合型慢病毒系统中各载体的比例是经过优化的，极大地提高了病毒包装所需蛋白的表达量。
其次，引入了Tet-Off转录激活子(tTA)，形成病毒包装蛋白的级联放大表达效应，极大地提高了病毒包装蛋白的表达量。
第三，百恩维生物拥有转染效率极高的转染试剂，可以高效地转染Lenti-X 293T包装细胞系，制备出高滴度的慢病毒颗粒。
本系统可以与任何慢病毒表达载体相兼容。
非整合型慢病毒主要优点：• 高效的基因转导，不会插入宿主基因组• 插入突变的风险低• 采用VSV-G包膜，宿主范围广泛• 分裂细胞中的瞬时表达，非分裂细胞中的稳定表达• 与任何慢病毒载体配套使用，产生非整合型的慢病毒• 产生高滴度慢病毒简而言之，非整合型慢病毒高表达目的基因，又不会引起突变，是一种非常有效的基因研究工具。
作为病毒包装领域的佼佼者，百恩维生物还有逆转录病毒，是基因研究技术服务的理想选择。
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A. Automatic Plate Washing..................................................................................................................... 7 B. Manual Plate Washing......................................................................................................................... 7 VII. Lenti-X p24 Assay Procedures................................................................................................ 8 A. Specimen Collection and Storage........................................................................................................ 8 B. Wash Buffer Preparation...................................................................................................................... 8 C. Protocol: Preparing Dilutions for the p24 Standard Curve.................................................................. 8 D. Protocol: Assaying Your Lentiviral Supernatants................................................................................. 9 VIII. Interpretation of Results...................................................................................................... 11 A. Assay Validation................................................................................................................................ 11 B. Determining Your Virus Titer............................................................................................................ 11 IX. Assay Troubleshooting.......................................................................................................... 12
List of Figures Figure 1. Lentiviral p24 is a virus core/capsid protein................................................................................. 3 Figure 2. The Lenti-X p24 Rapid Titer Kit................................................................................................. 3 Figure 3. Lenti-X p24 Rapid Titer Kit Standard Curve............................................................................ 10
Streptavidin-HRP Anti p24-Biotin p24 Anti-p24 coated well
Figure 2. The Lenti-X p24 Rapid Titer Kit. Lentiviral (HIV-1) p24 core protein in packaging cell supernatants is bound to wells of a microtiter plate coated with HIV-1 p24 capture antibody. The presence of bound p24 in the wells is detected using a biotinylated secondary anti-p24 antibody, a streptavidin-horseradish peroxidase conjugate, and a colorproducing substrate. Quantitation is performed by comparing test samples to a p24 standard curve.
The Lenti-X p24 Rapid Titer Kit allows you to quickly determine the titer of any HIV-1-based lentiviral supernatant using standard ELISA methods. The wells of the included microtiter plate (12 x 8-well strips) are coated with an antiHIV-1 p24 capture antibody, which quantitatively binds the HIV-1 p24 in your test samples (p24 is an abundant HIV-1 virus core/capsid protein [Figure 1]). Specifically-bound p24 is detected in a typical “sandwich” ELISA format using a biotinylated anti-p24 secondary antibody, a streptavidin-HRP conjugate, and a color producing substrate (Figure 2). Color intensity is measured spectrophotometrically to indicate the level of p24 in the samples, which can then be precisely quantified against a p24 standard curve. p24 values can then be correlated to virus titer of packaging cell supernatants.
Cat. No. 632200 PT5002-1 (PR932742) Published 5 May 2009
Lenti-X™ p24 Rapid Titer Kit User Ma. Principle of the Lenti-X p24 Rapid Titer Assay...................................................................... 3 II. List of Components................................................................................................................ 4 III. Storage and Stability.............................................................................................................. 4 IV. Safety Guidelines for Working with Lentiviruses................................................................. 5 V. Additional Materials Required............................................................................................... 6 VI. Assay Techniques & Microtiter Plate Washing...................................................................... 7
Figure 1. Lentiviral p24 is a virus core/capsid protein. The HIV-1 gag gene encodes p24, which is the major lentiviral capsid protein. The lysis of viral particles is necessary to generate soluble p24 which can then be measured by the Lenti-X p24 Rapid Titer Kit.