Lenti-X —Clone, Purify And Package - Takara.co.kr

Lenti-X —Clone, Purify and PackageOur cutting-edge research tools let you: Seamlessly clone into any vector Purify constructs quickly and efficiently Obtain exceptionally high lentiviral titers( 108 IFU/ml)

CloneGet the exact clone you want the first time!In-FusionTM Advantage PCR Cloning Kit Fast, easy, directional cloning of ANY insert into ANY vector Clone SEVERAL PCR products simultaneously Create seamless fusions with no unwanted bases added Get up to 100% cloning efficiency Clone in only 30 minIn-Fusion is a universal method for rapid, ligase- and restriction enzyme-independent, directional cloning ofPCR products. One or more PCR products can be inserted into ANY location in ANY linearized vector, providedthat the 15 bases at the ends of the PCR-amplified insert are complementary to the bases at the ends of thevector. The highly efficient In-Fusion enzyme facilitates rapid and precise cloning of PCR products, making itthe cloning system of choice for all cloning applications, including high-throughput.Superior cloning efficiency over a broadrange of insert sizes.Obtain 90% cloning efficiency, whetheryour vector has blunt or sticky ends.Side-by-side comparisons of the In-Fusion AdvantageCloning System and other vendors’ PCR cloningkits revealed In-Fusion’s superior ability to produceabundant clones regardless of insert length (see graphsbelow). Moreover, In-Fusion’s higher cloning efficiencycorresponded to a higher number of correct clones.The In-Fusion Advantage Cloning System was usedto clone a 2 kb fragment into three linearized vectorscontaining either blunt ends, 5’ and 3’ overhangs, or 3’overhangs. Random colony screening confirmed highcloning efficiencies with all three linearized vectors(see below), showing that In-Fusion works equally wellregardless of the nature of the vector ends.Blunt Ended Vector InsertCloning Efficiency for 0.5-4kb InsertsM6,000MTotal Colony NumberColonies that containcorrect insert4,0002,000Vector with 5' and 3' Overhangs InsertM0In-FusionProduct TProduct SCloning Efficiency for Inserts 4-6 kb3,000Total Colony NumberColonies that containcorrect insert2,0001,0000In-FusionProduct TProduct SVector 3' Overhangs InsertM

PurifyUltra-fast, high yield, endotoxin-free plasmid prepsNucleoBond Xtra Midi/Maxi EF Xtra fast no additional incubation for endotoxin removal 100 min (Midi), 45 min (Midi Plus), 103 min (Maxi) & 48 min (Maxi Plus) Xtra efficient endotoxin removal obtain DNA with less than 0.05 EU/µg Xtra high yields 250 µg for Midi & 1,000 µg for MaxiThe NucleoBond Xtra Midi EF and Maxi EF kits offer the superior performance of anion exchangetechnology plus optimized buffers for endotoxin-free plasmid DNA purification. DNA is purified andendotoxin removed in a simple procedure that requires only one wash step more than standard plasmidpreps, with no additional incubation time for endotoxin removal.Superior yields are obtained in less timewith NucleoBond Xtra EFDNA purified with NucleoBond Xtra EF issuitable for lentivirus production.Two NucleoBond Xtra Maxi EF Kits and a competitor kitwere used (according to each manufacturer’s protocol) toisolate a high copy number plasmid from the maximumculture volume recommended. Plasmid yields weredetermined after DNA precipitation (see table below).Endotoxin levels, determined using the LAL test (LimulusAmebocyte Lysate Pyrochrome; Lonza/Cambrex/BioWhittaker), were shown to be less than 0.01 EU/μg DNAfor all preps. NucleoBond Xtra EF saved up to 68% moretime and provided up to 100% higher yields compared toCompetitor Q’s kit.Expression vectors pLVX-AcGFP1-Actin and pLVX-DsRedMonomer-Nuc were purified with NucleoBond Xtra EF andpackaged into lentiviral particles using Clontech’s Lenti-X HT Packaging System. The resulting lentiviral particles(which averaged 1.9x108 IFU/ml) were used to cotransducehuman neural progenitor cells. The cells were visualized byfluorescence microscopy 48 hr after infection (see below).More than 99% of the transduced cells expressed AcGFPand DsRed.Table I: NucleoBond Xtra Maxi EF Performance Compared toa Competitor Endotoxin-Free Anion Exchange-Based KitMaxi KitsPlasmidYield (µg)Prep. Time(min)EndotoxinLevel (EU/µg)NucleoBond XtraMaxi Plus EF1,08048 0.01NucleoBond XtraMaxi EF1,200103 0.01Competitor Q600151 0.01

PackageGene of InterestPackage5' LTRΨPCMV IERREGenerate up to 25 times more lentivirus than otherpopular systemscPPTPPGKPuroRMCSWPRE3' LTRtTALentiphos HTtransfectionLenti-X HT Packaging Systems provide:Lenti-X HTPackaging Mix 108 IFU/ml A high safety profile Compatibility with any lentiviral vector293T cells1. Collect virus after 48 hr2. Transduce host cellsConcentrateIncrease your viral titer 100-fold withoutultracentrifugationAdd Lenti-X Concentratorto clarified viral supernatantLenti-X Concentrator highlights: Simple protocol: mix and spin Easily scalable: efficient concentration from smallor large volumes of lentiviral supernatant No ultracentrifugation required No specialized equipment required Store at –80 C Titrate Infect targetsIncubate at 4 Cfor 30 minto overnightCentrifugeat 1,500 x g at 4 Cfor 45 minCollect pelletand resuspendin 1/10 to 1/100original volumePurifyPurifying virus is now as simple as purifying plasmid DNALenti-X Maxi Purification Kit—high yield & purity: For more efficient and consistent transductions, and healthier cells Efficiently removes transduction inhibitors and cytotoxiccontaminants Concentrates virus up to 10-fold Simple and gentle gravity-flow protocolBindWashEluteTitrateChoose from p24 ELISA or qRT-PCR-based kitsStreptavidin HRPAnti-p24 (biotinylated)Lentiviral p24Lenti-X p24 Rapid Titer Kit: Fast and easy ELISA-based titration protocolAnti-p24-coated wellLentiviralpackaging cells Simply collect supernatant, lyse, bind, wash, and detect Correlate p24 content to virusAnti-p24-coated plate(12 x 8-well strips)Want to titrate virus by qPCR? The Lenti-X qRT-PCR Titration Kit lets you determine viral titers in only 4 hours.

Clone—we offer a wide variety of products for cloning, such as:Lenti-X Vectors Constitutive cDNA Expression Inducible cDNA Expression—based on Tet-On and Tet-Off Advanced shRNA Expression for RNAi ProteoTuner Protein Control—rapidly and reversibly control the level of yourprotein of interest Fluorescent Tag Expression—green and red fluorescence Bicistronic Vectors Reporter ExpressionInsert Amplification/Vector Linearization Advantage HD Polymerase Mix—for ultra-high-fidelity PCRPCR Product Preparation NucleoSpin Extract II—for PCR clean-up or gel extraction Cloning Enhancer—no need to perform PCR purification before cloningCloning Kits In-Fusion Advantage PCR Cloning Kits—get the exact clone you want thefirst time! In-Fusion Dry-Down PCR Cloning Kits—ready-to-use format for low-, medium-,and high-throughput applications.Purify—with NucleoBond Xtra EF Kits:Plasmid PurificationP NucleoBond Xtra Midi/Maxi—for fast purification when downstreamapplications involve sensitive cells. NucleoBond Xtra Midi/Maxi Plus—for ultra-fast purification whendownstream applications involve sensitive cells. NucleoBond Xtra Midi/Maxi EF—for fast purification when downstreamapplications involve highly sensitive cells. NucleoBond Xtra Midi/Maxi EF—for ultra-fast purification whendownstream applications involve highly sensitive cells.Package, Concentrate and Titrate—with Lenti-X Products:Lentiviral Packaging Systems Lenti-X HT Packaging System—generates up to 25 times more lentivirus Lenti-X HT Ecotropic Packaging System—for a murine-restricted tropismLentivirus Concentrator Lenti-X Concentrator—increase your viral titer 100 fold, withoutultracentrifugationLentivirus Titration Kits Lenti-X qRT-PCR Titration Kit—harvest, titrate, and infect in a single day! Lenti-X p24 Rapid Titer Kit—ELISA-based titration protocol

Xfect고객지원센터 (학술/제품문의) 02-2081-2510www.takara.co.kr support@takara.co.kr본 사대전지사Tel. 02-2081-2510 Tel. 042-828-6525Fax. 02-2081-2500 Fax. 042-828-6526

Purify Xtra fast † no additional incubation for endotoxin removal † 100 min (Midi), 45 min (Midi Plus), 103 min (Maxi) & 48 min (Maxi Plus) Xtra effi cient endotoxin removal † obtain DNA with less than 0.05 EU/µg Xtra high yields † 250 µg for Midi & 1,000 µg for Maxi DNA purifi ed with NucleoBond Xtra EF is suitable for lentivirus production.