Xfect转染试剂操作中文手册(clontech)

各种转染试剂的中文转染方法FuGENE6（Roche）转染步骤：转染前一天将细胞分至培养板，转染当天细胞应50-80％融合。

将细胞以1-3×105/2 ml接种于6孔板后孵育过夜将达到如此密度。

将FuGENE6 Reagent在室温孵育10-15分钟。

使用之前将FuGENE6颠倒混匀一下。

1. 在PCR管中加入不含血清和双抗的营养液以稀释FuGENE6，直至总体积到100 ul。

2. 将3-6 ul FuGENE6 Reagent直接加入营养液，轻弹管壁混合。

3. 加入1-2 ug的DNA溶液（0.02－2.0 ug/ul），轻弹管壁混合。

4. 室温孵育20分钟。

5. 将6孔板中的旧营养液吸出，加入约1 ml不含血清和双抗的营养液洗涤一次，再加入2 ml不含血清和双抗的营养液。

6. 将转染复合物加入细胞，混匀使之均匀分布。

7. 3-8小时后，加入血清或换成含血清的营养液。

Lipofectamine 2000(Invitrogen)转染试剂转染步骤（6孔板）：1. 转染前一天，胰酶消化细胞并计数，细胞铺板，使其在转染日密度为90-95％。

细胞铺板在2 ml含血清，不含抗生素的正常生长的培养基中。

2. 对于每孔细胞，使用250 ul无血清培养基（如OPTI-MEM I培养基）稀释4.0 ugDNA，轻轻混匀。

3. 使用前将Lipofectamine 2000转染试剂轻轻混匀，用250 ul无血清培养基（如OPTI-MEM I培养基）稀释10 ul Lipofectamine 2000转染试剂，轻轻混匀。

Lipofectamine 2000稀释后，在5分钟内同稀释的DNA混合（<30分钟）。

NOTE：若使用DMEM培养基，则需在5分钟内同稀释的DNA混合。

4. 混合稀释的DNA（第二步）和稀释的Lipofectamine 2000（第三步）。

室温放置20分钟。

5. （optional）将6孔板中的旧营养液吸出，用无血清培养基清洗两次。

罗⽒转染试剂说明书X-tremeGENE HP DNA Transfection ReagentFor transient and stable transfection of eukaryotic cellsVersion 05Content version: May 2011Cat. No. 06 365 752 001Cat. No. 06 366 244 001Cat. No. 06 366 236 001Cat. No. 06 366 546 001Trial-pack 0.4 ml 1 ml 5 ×1 mlStore at –15 to –25°C1.What this Product DoesNumber of TestsUsing the standard procedure, 1 ml of X-tremeGENE HP DNA Trans-fection Reagent can be used to perform up to 10,000 transfections in 96-well plates.FormulationX-tremeGENE HP DNA Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0.2 ?m pore size membrane, and packaged in glass vials. It does not contain any ingredients of human or animal origin.Storage and StabilityStore X-tremeGENE HP DNA Transfection Reagent at –15 to –25°C, with the lid tightly closed. The reagent is stable until the expiration date printed on the label when stored under these conditions.L X-tremeGENE HP DNA Transfection Reagent remains fully func-tional even after repeated opening of the vial (at least five times over a two-month period), as long as the vial is tightly recapped and stored at -15 to -25°C.L Note that the shipping temperature of this product is differentfrom the storage temperature. These different temperatures will not affect product performance or product stability.Special HandlingN After removing the amount required, tightly close the vial with thelid immediately after use.N Always bring the vial to +15 to +25°C and mix X-tremeGENE HPDNA Transfection Reagent prior to removing the amount required vortexing for one second.N Do not aliquot X-tremeGENE HP DNA Transfection Reagent; storein the original glass vials.N Minimize the contact of undiluted X-tremeGENE HP DNA Trans-fection Reagent with plastic surfaces.N For use, the minimum amount of X-tremeGENE HP DNA Transfec-tion Reagent: DNA complex is 100 µl. Complex formation at lower volumes can significantly decrease transfection efficiency.N Do not use tubes or microplates made of polystyrene forX-tremeGENE HP Transfection Reagent : DNA complex prepara-tion. When not able to avoid polystyrene materials, make certain to pipet the transfection reagent directly into the serum-free medium (e.g., Opti-Mem).N Do not use siliconized pipette tips or tubes.Additional reagents and equipment required to perform transfection assays using X-tremeGENE HP DNA Transfection Reagent include:?Standard Laboratory Equipment .Standard cell culture equipment (e.g., biohazard hoods, incuba-tors)Standard pipettes and micropipettes Vortex mixerFor Plasmid PreparationPurified plasmid stock (0.1 – 2.0 µg/µl) in sterile TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer or sterile waterGenopure Plasmid Midi Kit* or Genopure Plasmid Maxi Kit* to prepare plasmidFor Verification of Vector Function Assay appropriately for transfected geneG-418 Solution* or Hygromycin B* (optional for stable transfec-tion experiments)For Transfection-Complex FormationOpti-MEM I Reduced Serum Medium or serum-free medium Sterile polypropylene tubes or round-bottom 96-well plates Growing CellsSelect subconfluent cultures in log phase for preparation of cell culturesQuantify cell number to reproducibly plate the same number of cells ApplicationX-tremeGENE HP DNA Transfection Reagent is a high performance transfection reagent, free of animal-derived components. Benefits of X-tremeGENE HP DNA Transfection Reagent include:Designed to transfect a broad range of eukaryotic cells, including insect cells, many cell lines not transfected well by other reagents, and hard-to-transfect cell lines (e.g., HT-1080, K-562, HepG2).Can be successfully used in a variety of applications, such as gene expression analysis and protein production using transiently trans-fected cells, generation of stable cell lines, expression of shRNA for gene knockdown studies, drug discovery programs, and target evaluation. Samples and detailed transfection protocols are avail-able at/doc/48879f4bad02de80d4d840ed.html .Produces minimal cytotoxicity or changes in morphology when ade-quate numbers of cells are transfected, eliminating the requirement to change media after adding the transfection complex.?Suitable for transient and stable transfection. Functions very well in the presence or absence of serum.For life science research only.Not for use in diagnostic procedures.Print2.How to Use this Product2.1Before You BeginRequired Amount of X-tremeGENE HP DNA Transfection ReagentTo optimize, first transfect a monolayer of cells that is 70 - 90% conflu-ent, using 1:1, 2:1, 3:1 and 4:1 ratios of microliter (?l) X-tremeGENE HP DNA Transfection Reagent to microgram (?g) DNA. A ratio of 3:1 of microliter (?l) X-tremeGENE HP DNA Transfection Reagent to micro-gram (?g) DNA has been shown to be optimal for many cell types.L Lower cell confluencies have also been tested successfully.The recommended starting concentration is a 3:1. For most cell types, these X-tremeGENE HP DNA Transfection Reagent to DNA ratios pro-vide excellent transfection efficiency.L Further optimization may increase transfection efficiency in your particular application. In addition to varying the ratio, other param-eters may also be evaluated, such as the amount of transfection complex added. For additional optimization guidelines, see Section 3, Troubleshooting and visit /doc/48879f4bad02de80d4d840ed.html .For best results, accurately determine the plasmid DNA concentra-tion using 260-nm absorption; estimates of DNA by measuring gel band density are not recommended. Determine DNA purity using a 260 nm/280 nm ratio (the optimal ratio is 1.8).Prepare the plasmid DNA solution using sterile TE (Tris/EDTA) buf-fer or sterile water at a concentration of 0.1 to 2.0 µg/µl. Use high quality DNA preparation kits to obtain endotoxin-free DNA.Cell Culture ConditionsMinimize intra- and inter-experimental variance in transfection effi-ciency using cells that are regularly passaged, proliferating well in a log-growth phase, and plated at a consistent density.For best results, accurately quantify cell concentration using a hematocytometer or automated system.Cells must be healthy and free of Mycoplasma.Cells should have a low passage number to achieve best results.Other Media AdditivesIn some cell types, antimicrobial agents (e.g., antibiotics and fungi-cides) commonly included in cell-culture media may adversely affect the transfection efficiency of X-tremeGENE HP DNA Transfection Reagent. If possible, exclude additives in initial experiments. Once high-efficiency conditions have been established, these components can be added back while monitoring transfection results. Cell growth and/or transfection efficiency may be affected by variations in serum quality and medium formulations.Verification of Vector FunctionOptimize transfection conditions using a known positive-control reporter gene construct before transfecting cells with a new vector construct:Determine transfection efficiency using a reporter gene assay, such as -Gal*, Luciferase*, or SEAP*.Sequence flanking vector insert regions to verify the integrity of your new construct.2.2Preparation of Cells for TransfectionAdherent Cells: Plate cells approximately 24 hours before transfec-tion making sure cells are at the optimal concentration in the appropri-ate cell culture vessel.Suspension Cells: Plate freshly passaged cells at optimal concentra-tion.2.3 Transfection ProcedureAllow X-tremeGENE HP DNA Transfection Reagent, DNA and diluent to equilibrate to +15 to +25°C. Briefly vortex theX-tremeGENE HP DNA Transfection Reagent vial.Dilute DNA with appropriate diluent (e.g., serum-free medium) to a final concentration of 1 µg plasmid DNA /100 µl medium (0.01 µg/µl). Mix gently.Place 100 µl of diluent, containing 1 µg DNA into each of four sterile tubes labeled 1:1, 2:1, 3:1, and 4:1.N Use a minimum of 100 µl of diluent. Lower volumes may significantly decrease transfection efficiency.L Use sterile tubes or tissue culture treated round-bottom, 96-well plates to produce the complex.Pipet the X-tremeGENE HP DNA Transfection Reagent (1, 2, 3, or 4 µl) directly into the medium containing the diluted DNA without coming into contact with the walls of the plastic tubes.Mix gently.N To avoid adversely affecting transfection efficiency, do not allow undiluted X-tremeGENE HP DNA Transfection Reagent to come into contact with plastic surfaces. Do notIncubate the transfection reagent:DNA complex for 15 min-utes at +15 to +25°C.L Some ratios and cell types may required longer incubation (up to 30 min). Determine this for your particular cell lineand the ratio used.Remove the culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection com-plex to the cells in a dropwise manner.L See Table 1 to determine component amounts corre-sponding to the surface area of the cell culture vesselused.Gently shake or swirl the wells or flasks to ensure even distri-bution over the entire plate surface. If available, use a rotatingplatform shaker for 30 seconds at low speed for mixing96-well plates.Once the transfection reagent: DNA complex has been addedto the cells, there is no need to replace with fresh medium (asmay be necessary with other transfection reagents)Following transfection, incubate cells for 18 – 72 hours before measuring protein expression. The duration of incubation will depend on many factors, including the transfected vector con-struct, the cell type being transfected, the cell medium, celldensity, and the type of protein being expressed. After theincubation period, measure protein expression using an assayappropriate for your system.Notes:L As with any experiment, include appropriate controls. Prepare cul-ture wells with cells that remain untransfected, cells with transfec-tion reagent alone, and cells with DNA alone.L For stable transfection experiments, the complex-containing medium should be left unchanged until the cells are passaged. At that time, include appropriate selection antibiotics (e.g., G 418 Solution or Hygromycin B).L To prepare transfection complexes for different-sized containers or parallel experiments, adjust component amounts correspond-ing to the surface area of the cell culture vessel used (see Table 1).L For ease-of-use when transfecting small volumes into 96-well plates containing 0.1 ml culture medium per well, prepare 100 µl of transfection complex, and then add 10 µl to each well (depend-ing on cell type).L The optimal ratio of transfection reagent to DNA, and the optimal total amount of complex, will depend on the cell line, cell density, day of assay, and gene expressed.L After performing the optimization experiment in which several dif-ferent ratios are tested, select a ratio in the middle of the plateau optimum for future experiments.Tab. 1: Guidelines for Preparing X-tremeGENE HP DNA Transfection Reagent: DNA Complex for Various Culture Vessel SizesCulture vessel Surface Areamedium (ml) Suggested amount of100 µl transfection complexto add toeach well (µl) DNA (µg)using 1:1or 4:1 RatioFinal amountof X-tremeGENE HP DNA Transfection Reagent (µl) using 1:1 Ratio Final amountof X-tremeGENE HP DNA Transfection Reagent (µl) using 4:1 Ratio 96-well plate(1 well)0.30.1100.10.10.4 48-well plate(1 well)1.00.3300.30.3 1.2 24-well plate(1 well)1.90.5500.50.52 12-well plate(1 well)(1 well)9.42200228 60-mm dish2155005520 10-cm dish55101000101040 T-25 flask2566006624 T-75 flask75202000202080 2.4TroubleshootingObservation Possible Cause RecommendationLow Transfection Efficiency Suboptimal X-tremeGENE HP DNA Transfec-tion Reagent : DNA ratioTitrate optimal X-tremeGENE HP DNA Transfection Reagent : DNAratio. Refer to the text in Section 2.1 “Before you begin”.Insufficient number of cells Determine optimal cell density for each cell type. For most cell types, 70– 90% confluence at transfection is optimal.X-tremeGENE HP DNA Transfection Reagent :DNA complexes did not form wellPrepare complexes in serum-free medium (e.g., Opti-MEM).Do not use siliconized pipet tips or tubes.Do not aliquot the X-tremeGENE HP DNA Transfection Reagent. Incubation time of transfection Determine the optimal incubation time (18 - 72 h). Optimal for most celltypes and plasmids is 24 – 48h.Inhibition by media components Some media components (e.g., polyanions) may influence the transfec-tion.Low volume of X-tremeGENE HP DNA Trans-fection Reagent : DNA complexThe minimum amount of X-tremeGENE HP DNA Transfection Reagentto DNA complex is 100 µl. Complex formation at lower volumes may sig-nificantly decrease the transfection efficiency; refer to the text in Section1, “Special Handling”.High Cytotoxicity Cell density not optimal For each cell type, the optimal density should be determined. For mostcell types, 70 - 90 % confluence at transfection is recommended, butother confluencies may increase cell viability.Cells are cultured in serum-free medium Transfection using X-tremeGENE HP DNA Transfection Reagent in cells cultured in serum-free medium is possible, however, toxicity may behigher when serum is absent.X-tremeGENE HP DNA Transfection Reagent : DNA complexes and cells not mixed well Add X-tremeGENE HP DNA Transfection Reagent dropwise to the cells. Gently rock the dish/plate back and forth and from side to side to evenly distribute the complexes.Plasmid preparation contaminated with endo-toxinTransfected protein is cytotoxic or is produced at high levels Reduced viability or slow growth rates may be due to high levels of pro-tein expression, with cellular metabolism directed toward production of the heterologous protein. Note that the expressed protein may also be cytotoxic at the expressed levels.Too much transfection complex for number of cells Increase the number of plated cells, and/or decrease the total amount of complex added to the cells.3.Additional Information on This ProductQuality ControlEach lot of X-tremeGENE HP DNA Transfection Reagent is tested using established quality control procedures. Functional AnalysisCells are transfected with a reporter gene vector DNA using X-tremeGENE HP DNA Transfection Reagent (ratio 3:1 µl/µg DNA). Reporter gene activity is monitored by chemiluminescent detection. Using a standard curve analysis method, total amounts of recombinant protein per well are measured to ensure levels that are within specifi-cation.4.ResultsCHO-K1 cells were transfected with a GFP encoding pcDNA3.1 plas-mid containing a CMV promoter with two different transfection reagents. CHO-K1 cells were observed under fluorescence and bright field microscopy at 10× magnification. Pictures were obtained using the Cellavista System 24 hours after transfection.Fig. 1:X-tremeGENE HP DNA Transfection Reagent (1:1 ratio)Fig. 2:Competitor transfection reagent (2:1 ratio)5.Supplementary InformationConventionsIn this document, the following symbols are used to highlight impor-tant information:Symbol DescriptionL Information Note:Additional information about the current topic or proce-dure.N Important Note:Information critical to the success of the procedure or useof the product.Text ConventionsTo make information consistent and understandable, the following textText Convention UseNumbered instructionslabeled ?, ? etc.Steps in a procedure that must be performedin the order listed.Changes to Previous VersionEditorial changesOrdering InformationRoche Applied Science provides a large selection of reagents and sys-tems for life science research. For a complete overview of relatedproducts and manuals, visit and bookmark our home page,/doc/48879f4bad02de80d4d840ed.html , and our Special Interest Site on transfection, /doc/48879f4bad02de80d4d840ed.htmlAsterisk *Denotes a product available from Roche AppliedScience.Product Pack Size Cat. No.Apoptosis and Cell Death ProductsCell ProliferationReagent WST-125 ml (2,500 tests)8 ml (800 tests)11 644 807 001***********Cytotoxicity DetectionKit PLUS (LDH)1 kit 400 tests in 96 wells1 kit 2,000 tests in 96 wells**********************Gene Knockdown ReagentX-tremeGENE siRNATransfection Reagent1 ml (400 transfections in a24-well plate)Mycoplasma Detection ReagentsMycoplasma DetectionKit1 kit (25 tests)11 296 744 001Mycoplasma PCR ELISA1 kit (96 reactions)11 663 925 910 Plasmid Isolation ProductsGenopure Plasmid MidiKit1 kit (for up to 20 prepara-tions)***********Genopure Plasmid MaxiKit1 kit (for up to 10 prepara-tions)***********Protease Inhibitor T ablets and Lysis Reagents cOmplete20 tablets in glass vials3 x 20 tablets in glass vials20 tablets in EASYpacks11 697 498 00111 836 145 001***********cOmplete, EDTA-free20 tablets in a glass vial3 x 20 tablets in glass vials20 tablets in EASYpacks11 873 580 001**********************cOmplete Lysis-M (formammalian cell lysis)1 kit (200 ml lysis reagentand 20 complete ProteaseInhibitor Cocktail Tablets)Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim GermanyContact and SupportTo ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at:/doc/48879f4bad02de80d4d840ed.html /supportTo call, write, fax, or email us, visit the Roche Applied Science home page,/doc/48879f4bad02de80d4d840ed.html , and select your home country. Country- specific contact information will be displayed. Use the Product Search func-tion to find Pack Inserts and Material Safety Data Sheets. Regulatory DisclaimerFor life science research only.Not for use in diagnostic procedures.TrademarksCOMPLETE, GENOPURE, X-TREMEGENE, XCELLIGENCE, CASY, CEDEX, and CELLAVISTA are trademarks of Roche. E-PLATE and ACEA BIOSCIENCES are registered trademarks of ACEA Biosciences, Inc. in the US.Other brands or product names are trademarks of their respective holders.cOmplete Lysis-M,EDTA-free (for mammalian cell lysis) 1 kit (200 ml lysis reagent and 20 complete, EDTA-free Protease Inhibi-tor Cocktail Tablets)04 719 964 001Reporter Gene Assays CAT ELISA1 kit (192 tests)11 363 727 001-Gal Reporter Gene Assay,chemiluminescent 1 kit (500 assays, micro-plate format, 250 assays, tube format)11 758 241 001?-Gal ELISA 1 kit (192 tests)11 539 426 001hGH ELISA1 kit (192 tests)11 585 878 001Luciferase Reporter Gene Assay, high sensi-tivity200 assays 1,000 assays 11 669 893 00111 814 036 001SEAP Reporter Gene Assay,chemiluminescent 1 kit (500 assays, micro-plate format, or 250 assays, tube format)11 779 842 001Selection Antibiotics G-418 Solution 20 ml 100 ml 04 727 878 00104 727 894 001Hygromycin B1 g (20 ml)10 843 555 001Transfection Reagents X-tremeGENE 9 DNA Transfection Reagent0.4 ml 1 ml 5 x 1 ml 06 365 779 00106 365 787 00106 365 809 001Western Blotting ReagentsLumi-Light PLUS Western Blotting Kit(Mouse/Rabbit)1 kitLumi-Light PLUS Western Blotting Substrate100 ml(1,000 cm 2 membrane)12 015 196 001PVDF Western Blotting Membranes 1 roll(30 cm × 3.00 m)03 010 040 001Western Blocking Reagent, Solution100 ml(10 blots, 100 cm 2)6 × 100 ml(60 blots, 100 cm 2)11 921 673 00111 921 681 001Cellular Analysis RTCA Analyzer 05 228 972 001RTCA SP Station 05 229 057 001RTCA MP Station 05 331 625 001RTCA Control Unit 1.105 454 417 001E-Plate 96 6 Units 6 x 6 Units 05 232 368 001 05 232 376 001E-Plate VIEW 96 6 Units 6 x 6 Units06 472 451 001 06 472 460 001Cellavista BasicMagnification: 4x, 10x Illumination: Brightfield only***********Cellavista Medium Magnification: 4x, 10x, 20xIllumination: Brightfield and Fluorescence, UV, Blue, Green***********Product Pack Size Cat. No.Cellavista High End Magnification: 2x, 4x, 10x, 20x, 40xIllumination: Brightfield and Fluorescence,UV, Blue, Cyan, Green, Amber, Red***********Cedex XS Analyzer with Control Unit 05 926 432 001Cedex Smart Slide package15 x 8measurements05 650 801 001CASY Model TT 45, 60, 150 µm***********ProductPack Size Cat. No.。

Tet-On® 3G 诱导表达系统实验流程一、测验共转染pTRE3G-GOI 载体和pCMV-Tet3G (比例1:4) HeLa 或HEK 293，或靶细胞。

Dox诱导目的表达之后，选择合适的特异基因分析方法进行分析目的基因的表达情况，例如：Western blotNorthern blotqRT-PCR基因特异性功能分析或者，将pTRE3G-GOI载体转染Tet-On 3G cell line，来考察目的基因的表达情况。

1. 使用Xfect转染试剂将调节载体和应答载体共转染靶细胞(在一个6-well板中进行)，参照Xfect 转染试剂使用手册(请登录/manuals下载说明书)。

每孔使用1 μg pCMV-Tet3G和4 μg pTRE3G-GOI。

建议设置实验组和阴性对照组：3个孔中添加100–1,000 ng/ml Dox，另外3个孔孔不添加Dox。

Wells 1 & 2: 1 μg pCMV-Tet3G and 4 μg pTRE3G-GOI (no Dox)Wells 3 & 4: 1 μg pCMV-Tet3G and 4 μg pTRE3G-GOI (100–1,000 ng/ml Dox)Well 5: 1 μg pCMV-Tet3G and 4 μg pTRE3G empty (no Dox)Well 6: 1 μg pCMV-Tet3G and 4 μg pTRE3G empty (100–1,000 ng/ml Dox)Figure 4.调节载体和应答载体转染六孔板中的靶细胞2. 24 hr后，分别收集每个孔中的细胞团，根据GOI选择合适的方法比较诱导后的GOI表达水平和未经诱导的GOI表达水平。

注意: 由于瞬时转染后的细胞比稳定转染的细胞内包含更多拷贝的TRE元件拷，瞬时转染实验中的基因诱导倍数比稳定转染实验的低，瞬时转染实验中的诱导倍数只能有10-100倍。

EndoFectin™-CHO 转染试剂■ 产品概述：EndoFectin™ CHO 转染试剂是一种具有专利的阳离子聚合物试剂，它能与核酸形成复合物，并使该复合物进入哺乳动物细胞。

EndoFectin™ CHO 转染试剂专为转染CHO 细胞，并构建稳定的细胞系而设计。

即使在有血清存在的情况下，它仍然能高效的将核酸导入细胞。

GeneCopoeia 公司提供的 EndoFectin™ CHO 转染试剂有如下优点：• 优越的转染效率• 重组蛋白的高表达水平• 与含血清的培养基相兼容• 低细胞毒性• 易于操作■ 成分及储存条件：• 每管含有经过滤除菌的EndoFectin™ CHO 转染试剂• EndoFectin™ CHO 转染试剂 可于常温下运输。

4-8℃密闭保存。

该试剂在4-8℃的条件下，可保持稳定至少12个月。

■ 质量控制：每批EndoFectin™ CHO 均经过转染测试。

我们将eGFP 表达质粒(GeneCopoeia Catalog No. EX-EGFP-Lv01)用EndoFectin™ CHO 转染试剂转入subconfluent HEK-293 细胞。

转染16小时后，超过95%的细胞表达eGFP 。

■ 实验开始前的注意事项：质粒的质量：请务必使用高质量转染级无内毒素的质粒。

通过260nm 光吸收测定DNA 浓度，260nm/280nm 比值确定DNA 纯度（比值应该在1.8~2.0的范围之内）。

如有可能，请通过琼脂糖凝胶电泳检测质粒的完整性。

细胞的条件：使用适当保存和经常传代的健康细胞。

确保培养基没有被细菌，真菌或支原体污染。

如果细胞是近期复苏的液氮冻存细胞，请在转染前至少传代两次。

■ 瞬时转染方法：1. 接种细胞1转染前一天，用胰酶消化细胞并计数。

调整细胞浓度，将细胞铺入细胞培养的器皿，总体积如表1所示。

每个孔置入的细胞量应能使转染时细胞汇合度达到70~80%。

2. 准备DNA/EndoFectin™复合物GeneCopoeia Inc.19520 Amaranth DriveGermantown, Maryland 20874USATel: 301-515-6982; 1-866-360-9531Fax: 301-515-6983Web: GeneCopoeiaTMExpressway to Discovery用于转染核酸到哺乳动物细胞产品套装编号： Z0103储存条件：4℃－8℃保存 产品编号Z01030A Z01030BZ01030C （A*5）包装规格1mL 0.5mL 5 mL地址: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，510663客服电话*************电子信箱:********************网址:该产品仅限于实验科学研究用，若有任何单位或个人将该产品用于临床诊断、治疗等其他国家专门规定的特殊用途，本公司概不承担任何责任。

Lipofectamine TM 2000CAT. NO. 11668-027 Size:CAT. NO. 11668-019 Size: ml4℃储存（不要冻存）说明：Lipofectamin TM 2000是核酸（DNA或RNA）转染真核细胞的一个专用的试剂盒，其有如下优点：对各种细胞及细胞板（如96孔板）都有高的转染效率，在的细胞系数据库中有各种细胞转染成功的实例。

在含有或是不含有血清的培养基中，DNA- Lipofectamin TM 2000复合物能够直接加给细胞。

在转染之后不需要除去复合物以及添加或是更换培养液，但在培养4-6小时后需要除去复合物。

关于转染的一些重要建议：1.不要用即将要介绍的转染程序进行RNAi的转染实验。

在上有转染步骤，登陆后点击说明。

2.对于大多数细胞系，转染复合物中DNA(μg)与Lipofectamine TM 2000(μl)的比例在1：2到1：3之间，最好达到最优化的比例。

注意：在混合之前，我们建议用Opti-MEM I 低血清培养基（Cat: ）（reduced serum medium）稀释Lipofectamine TM 2000和DNA.3.为了实现高的转染效率、高的目的基因表达水平以及低水平细胞毒效应，受体细胞最好达到高的浓度：在转染时，细胞的培养液的混浊度建议为90%-95%并最优化混浊度。

此外，在实验过程中保证相同的接种条件。

4.为避免细胞死亡，在培养基中不要加抗生素。

5.由于一些无血清复合物（如CD239、SFM II、VP-SFM）会抑制阳离子脂质体介导的转染，因此有必要检测一下无血清培养基和Lipofectamine TM 2000的相容性。

转染步骤（用于DNA）：按照如下步骤在24孔板中转染哺乳动物细胞。

对于其它种类细胞板请参照转染量度标准。

步骤中均按照一个细胞孔的量给出质量和体积。

1.贴壁细胞：转染的前一天，在500μl无抗生素培养基中接种×105个细胞，以保证在转染时候细胞的混浊度达到90%-95%。

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。

2016‐08‐15I. 司诺L灯头图示2016‐08‐152016‐08‐15II, 开始1. 打开灯头保护盖 (取走灯管前的保护海绵, 另确定灯管正确固定在插座上)3. 安装电池至灯头上。

2. 按一下电池后方的 “on/check” 按钮测试电池的电量。

4. 启动开关按钮 (在底部)2016‐08‐15III. 操作按钮按钮 /标示功能旋转按钮 (1)启动或关闭灯头（备用模式）。

按下”旋转按钮”维持3秒钟可启动或关闭灯头至备用模式。

轻按”旋转按钮”可进入菜单选项介面。

有关详细说明请参照第五章。

进入菜单模式后” 旋转按钮” 底部的蓝色指示灯会闪烁。

在操作模式下：转动”旋转按钮”：可调整灯头的输出功率。

反时钟方向转动可调减输出功率。

顺时钟方向转动可增加输出功率。

慢速转动可按1/10的功率调节，而快速转动可按1级光功率调节。

数字显示屏 (2) 显示输出功率或菜单的选取。

Mod / Eco 造型灯及省电模式开关 (3) 轻按启动或关闭造型灯。

长按按钮可改选为正常或省电模式。

Test (4)测试键当”旋动按钮”底部的蓝色灯亮着，表示灯头已充电及准备就绪。

可按测试键引发灯头作测试。

长按按钮可把灯头的设定回到默认设定Speed (5) 高速模式提升回电速度及闪速。

详细内容请参照第7章。

WiFi (6)启动或关闭wifi 接连功能。

st/ch (8) 无线引闪频道设定可选取布朗RFS 无线引发的频道。

lamp (9) 灯头地址设定可选取布朗RFS 频道的灯头地址。

2016‐08‐15seq (10)连续频闪设定可设定连续频闪的次数。

beep (11) 声音提示启动或关闭准备就緖的声音提示功能。

sync (12) 同步引闪 可选择启动或关闭哪一项同步引闪模式。

（无线引发及光敏感应同步）同歩线插口/ USB插口 (13/14) 同步线插入口供同步引闪用 / USB 插囗供固体升级用开关按钮 (15)启动或关闭灯头电源。

Certificate of AnalysisTakara Bio USA, Inc.2560 Orchard Parkway, San Jose, CA 95131, USAU.S. Technical Support: *******************************United States/Canada 800.662.2566 Asia Pacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.565.6999Page 1 of 1(011623) Xfect™ RNA Transfection ReagentCatalog No. Amount Lot Number631450 1.2 ml Specified on product label. DescriptionXfect RNA Transfection Reagent is a complete system for highly efficient transfection of mammalian cells with all types of RNA, including messenger RNA (mRNA), small interfering RNA (siRNA), and single guide RNA (sgRNA). The protocol is simple, and transfections can be carried out entirely in the presence of serum. Xfect RNA Transfection Reagent creates biodegradable nanoparticles when combined with RNA, resulting in very low cytotoxicity.Package Contents• 2 x 600 µl Xfect RNA Transfection Polymer• 2 x 12 ml Xfect Reaction BufferStorage Conditions•Store Xfect RNA Transfection Polymer and Xfect Reaction Buffer at –20°C. Do not thaw until ready to use. Once thawed, store at 4°C for up to 12 months.NOTE: The Xfect RNA Transfection Polymer is a milky suspension and should be vortexed briefly prior to use to ensure that it is fully resuspended.Expiration Date•Specified on product label.Shipping Conditions•Dry iceProduct DocumentsDocuments for our products are available for download at /manualsThe following documents apply to this product:•Xfect RNA Transfection Reagent Protocol-At-A-GlanceQuality Control DataTransfection Test for Xfect RNA Transfection PolymerHeLa cells were transfected with 1 µg of GFP mRNA in one well of a 12-well plate, according to the protocol outlined in the Xfect RNA Transfection Reagent Protocol-At-A-Glance. 48 hours post-transfection, the transfection efficiency was determined via flow cytometry and found to be greater than 70%.Sterility TestThere was no evidence of bacterial or fungal growth after inoculation on blood agar, thioglycolate medium, or Sabouraud dextrose agar.It is certified that this product meets the above specifications, as reviewed and approved by the Quality Department.CATALOG NO.631450NOTICE TO PURCHASER:Our products are to be used for Research Use Only . They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide aservice to third parties without our prior written approval.Your use of this product is also subject to compliance with the licensing requirements, listed below if applicable, and described on the product´s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by these statements.STATEMENT 91This product is intended for in vitro research purposes only. It may not be used for (i) any human or veterinary use,including without limitation therapeutic and prophylactic use, (ii) any clinical use, including without limitation diagnostic and prognostic use, (iii) screening of chemical and/or biological compounds for the identification of pharmaceutically active agents (including but not limited to screening of small molecules), target validation,preclinical testing services, or drug development or (iv) any commercial purposes, including without limitation the performance of contract research or provision of services to a third party and the manufacture of products for general sale. Any use of this product for any of the abovementioned purposes requires a license from the Massachusetts Institute of Technology.TRADEMARKS:©2023 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.。

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。

ESCORT™V Transfection ReagentCatalog Number E9778Storage Temperature 2-8 °CTECHNICAL BULLETINProduct DescriptionESCORT V Transfection Reagent was developed for highly efficient stable and transient transfections of eukaryotic cells. It is a specially processed polyethyleneimine (PEI) optimized for use in serum-containing media. Transfection in serum-free media has been demonstrated, and Escort V can also be used for transfection experiments with siRNA.The reagent has been extensively tested and found to be highly efficient in many cell types including CHO, BHK, HeLa, HEK293, HEK-293T, Vero, F9, PC12, COS-1, COS-7, NIH 3T3, L929, Human Foreskin Fibroblasts (HFF) primary cells, and Bovine Aorta Endothelial cells (BAEC).ReagentsESCORT V Transfection Reagent,Catalog Number E0654 1.5 ml ESCORT V Transfection Buffer,Catalog Number E0529100 ml ESCORT V Transfection Reagent is provided at a concentration of 1.3 mg/mL. The reagent is sufficient for 700-1000 transfection experiments in 24-well plates.Precautions and DisclaimerThese products are for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Storage/StabilityStore at 2-8 °C.PROCEDUREThis protocol is optimized for use in 24-well plates. All volumes given are for a single reaction (single well) on a 24-well plate. If another format is desired, scale up or down accordingly (Please see Table I). Cell PreparationAdherent cells1.18-24 hours before transfection, seed the plate with5-7.5 x 104 cells/well in 0.5 ml of the appropriatemedium.2.30 minutes to 2 hours before adding thetransfection cocktail to the wells, replace the growth media in the wells with 0.5ml fresh mediacontaining serum (complete media).Cells in suspension1.Before transfection, pellet the cells and dilute to theappropriate concentration (0.5-1X106cells/ml) inthe standard growth medium for the cell line.2.Add 0.5 ml of the cell suspension to each well. Transfection with plasmid DNA in 24-well plateFor optimal transfection, the recommended ratio ofµg DNA/µL ESCORT V Transfection Reagent is 1:3.In some cases, transfection efficiency can be increased by using a different ratio. When changing the amount of ESCORT V Transfection Reagent (step 2), keep the amount of Transfection Buffer constant at 60 µL.1.In a sterile tube, combine 60 µl of ESCORT VTransfection Buffer and 0.7 µg of plasmid DNA.Mix the contents of the tube gently.2.In a separate tube, combine 60 µl of ESCORT VTransfection Buffer and 2.1 µl of Escort VTransfection Reagent. Mix gently.bine the DNA/buffer solution, Step 1, with theESCORT V reagent/buffer solution, Step 2, to make the transfection cocktail. Mix gently.4.Incubate at room temperature for15-20 minutes.The complex is stable for up to 2 hours at roomtemperature.5.Add the total volume of transfection cocktail directlyto the wells. Mix gently by rocking the plate.6.Incubate the transfected cells under standardconditions for 24-72 hours.2Stable Transfection1.Perform cell transfection using the appropriatevector. Follow the protocol above.2.Two days after transfection, replace the media withfresh media containing the appropriate selectionantibiotic.3.Maintain the cells in selective media for 2-3 weeksuntil stably transfected cells are clearly identified. Transfection with siRNA in a 6-well plateThis protocol is optimized for cells plated in a 6-well plate. Volumes indicated in the protocol are for one reaction (one well).1.Prepare 10 nmol siRNA stock solution (dilute withRNAse-free water).2.In a sterile tube, add 60 µL of ESCORT VTransfection Buffer and 9 µL of ESCORT VTransfection Reagent.3.Mix the contents of the tube gently and incubate for5 minutes at room temperature.4.In a second sterile tube, add 60 µL of serum-freemedium and 10 µL of10 nmol siRNA solution.bine the contents of both tubes and gently mix.6.Incubate for 15-20 minutes at room temperature toform the transfection complex.7.During incubation, remove culture media from thewells and add 0.8 ml of complete culture media(media with serum) to each well.8.Carefully add the transfection mix drop-wise to thecells in the well.9.Swirl the plate gently to ensure uniform distributionof the transfection complex.10.Incubate the cells at 37 °C in 5% CO2for 24-72hours before analysis.11.Change the media as required.Transfection with siRNA in a 96-well plateThis protocol is optimized for a 96-well plate. Volumes indicated in the protocol are for one reaction (one well).1.Prepare 100 pmol siRNA stock solution (dilute withRNAse-free water).2.In a sterile tube, add 25 µL of ESCORT VTransfection Buffer and 0.6 µL of ESCORT VTransfection Reagent.3.Mix the contents of the tube gently and incubate for5 minutes at room temperature. 4.In a second sterile tube, add 25 µL of serum-freemedia and 10 µL of the 100 pmol siRNA solution. bine the contents of both tubes and mixcarefully.6.Incubate for 10-15 minutes at room temperature toform the transfection complex.7.During incubation, remove the culture media fromthe wells and add 100 µL of complete culture media (media with serum) to each well.8.Carefully add the transfection mix drop-wise ontothe cells in the well.9.Swirl the plate gently to ensure uniform distributionof the transfection complex.10.Incubate the cells at 37 °C in 5% CO2for 24-72hours before expression analysis.11.Change the media as required.OPTIMIZING TRANSFECTIONFor some cell lines, ESCORT V Transfection Reagent may require optimization. When optimizing conditions, it is important to consider the following optimization factors:Volume of Transfection CocktailIn some cases, a different volume of the transfection mixture may increase transfection rates. For optimization, compare transfection performance when different volumes of transfection mixture are added to the wells (e.g., 75, 100, 120, and 150 µL/well).The Ratio of DNA/ESCORT VThe recommended ratio of µg DNA/ µl ESCORT V Transfection Reagent is 1:3. This ratio produces good results for most cell lines. In some cases, transfection efficiency can be increased by changing the ratio (e.g., 1:2 or 1:4).MediaIt is recommended to change growth media up to 2 hours before transfection. Use complete media (media supplemented with serum). If desired, media containing the transfection mixture can be substituted 4-6 hours after the initial addition without loss of transfection activity.Transfection without ESCORT V Transfection BufferIf use of the ESCORT V Transfection Buffer is not desired, prepare the transfection mixture in any standard media without serum and antibiotics. Follow the same volumes as in transfection protocol.3Cell DensityThe optimal number of cells to be plated depends on the specific cell line. A 40%-70% confluent cell layer at the time of transfection is suggested for best response. When required, cells can be transfected at very low cell densities such as 5-15% confluence.Incubation Time Post-TransfectionIncubation time depends on the cell line, the protein being expressed, as well as the vector construct.DNA QualityDNA quality is a critical factor for successful transfection. OD260/280should be ~1.8 or greater (1.85 is recommended). DNA should be free of endotoxin and other contaminants. RNA contamination does not prevent transfection; however, RNA substitutes DNA in the complex and may lead to an incorrect DNA concentration estimation. Taken together, these factors may greatly reduce transfection efficiency.DNA VectorThe expression of the transfected gene depends on the cell line, the promoter used, and the nature of the expressed protein.Condition of the CellsCells should be healthy, free of contamination, proliferating well and plated at an appropriate density. The Level of the Expressed GeneHigh level of expression of some proteins can be cytotoxic.Table I Scaling up/down4Troubleshooting GuideESCORT is a trademark of Sigma-Aldrich Biotechnology LPKH,PHC 03/07-1Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications.Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply.Please see reverse side of the invoice or packing slip.。