lipofectamine2000转染说明
LIPOFECTAMINE2000转染试剂转染步骤
LIPOFECTAMINE2000转染试剂转染步骤转染是指将外源DNA或RNA导入到目标细胞中的过程，LIPOFECTAMINE2000是一种常用的转染试剂。
下面是使用LIPOFECTAMINE2000进行转染的详细步骤：步骤一：细胞处理1.1培养要转染的细胞株，并确保细胞达到70%-80%的密度。
1.2使用无菌PBS洗涤细胞，将细胞悬浮于含有10%FBS的完全培养基中。
1.3通过计数细胞数来得到适当的细胞密度，以确保每个孔或皿中有足够的细胞进行转染。
步骤二：DNA/RNA和转染试剂的配制2.1在无菌离心管中配制DNA/RNA和转染试剂的混合液。
按照试剂的说明书中的推荐比例将DNA/RNA和转染试剂混合在一起，并使用无菌PBS 或者培养基和其它试剂进行稀释。
2.2轻轻摇晃混合液，避免产生气泡。
步骤三：转染3.1将配制好的转染混合液加入到每个孔或皿中，并轻轻摇晃培养皿/板使其均匀分布。
3.2将细胞和转染试剂混合液共孵育4-6小时，在37℃的CO2培养箱中进行转染反应。
转染时间可以根据目标细胞的特性进行调整。
步骤四：更换培养基4.14-6小时后，将转染混合液完全去除，并用预温热的完全培养基洗涤细胞，以去除未吸附的DNA/RNA和转染试剂。
4.2加入足够的完全培养基来覆盖细胞，尽量减少液体涡流，以避免对转染效率的不良影响。
步骤五：细胞培养和分析5.1将培养皿/板放回37℃的CO2培养箱中，并进行适当的培养条件。
5.2根据实验需要的时间点收集转染后的细胞进行后续的实验和分析。
需要注意的是，转染步骤中的各种参数（例如细胞密度、转染试剂的浓度和比例等）可能因不同的实验目的和目标细胞而有所不同。
因此，在具体操作中请参考所使用转染试剂和目标细胞的说明书，并根据实验需要进行相应的优化。
Lipofectamine2000转染说明书
Lipofectamine™ 2000 Transfection Reagent TABLE OF CONTENTSPRODUCT DESCRIPTIONSHIPPING CONDITIONSSTORAGE CONDITIONSSTABILITYQC SPECIFICATIONSPROTOCOL & APPLICATION NOTESRelative Surface Areas Of Tissue Culture VesselsSurface Areas Of Tissue Culture VesselsTubes Recommended For Use With Lipofectamine™ 2000Optimizing Plasmid DNA Transfections With Lipofectamine™ 2000Transfection ProtocolsCo-Transfection Of Sirna And Plasmid DNATransfection Of Fluorescently Labeled Oligos With Lipofectamine™ 2000Transfection Of Cells In 96-Well PlatesTransient Transfection Of Suspension CellsALTERNATE PRODUCTS & COMPATIBILITYPRODUCT DOCUMENTATIONREFERENCESPRODUCT NAME & CATALOG NUMBERSASSOCIATED PRODUCTSRELATED TECHNICAL SUPPORT NOTESPRODUCT DESCRIPTION(back to Table of Content)Lipofectamine™ 2000 Transfection Reagent is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells and provides the following advantages:Highest transfection efficiency in many cell types and formats (e.g. 96-well). Refer to the Cell Lines Database for a list of cell types successfully transfected. Detailed in-house transfection protocols are also available at this site whereavailable.DNA-Lipofectamine™ 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.Lipofectamine™ 2000 may be used in the following applications:Transient and stable transfection of adherent and suspension cellsHigh throughput transfectionsDelivery of Stealth RNAi and siRNA into cells For information on transfecting mammalian cells with short interfering RNAs (siRNA) for use in RNA interference (RNAi) studies, visit our RNAi Central web page at\rnai. Cell line-specific protocols are available under the Protocols tab.Lipofectamine™ 2000 gives superior transfection efficiency for the following cell lines:293F 293H BE(2)C (w/o serum)serum)(w/o(adherent) COS-1CHO-K1 CHO-SFibroblasts (w/o serum)HumanPrimaryCOS7-Lserum) HT-1080 MDCK(w/oHT-29SK-BR3serum) PC12MRC-5(w/o3T3NIHHepG2VeroLipofectamine™ 2000 CD is a 100% synthetic version of Lipofectamine™ 2000. Use it in the same way as Lipofectamine™ 2000, but be sure to use animal origin-free reagents.SHIPPING CONDITIONS(back to Table of Content)This kit is shipped on wet ice.STORAGE CONDITIONS(back to Table of Content)Lipofectamine™ 2000 Transfection Reagent should be stored at 40 C.STABILITY(back to Table of Content)The stability of Lipofectamine™ 2000 Transfection Reagent is guaranteed for 6 months when it has been stored as recommended.Lipofectamine™ 2000 Reagent should not be frozen.QC SPECIFICATIONS(back to Table of Content)Lipofectamine™ 2000 is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.PROTOCOL AND APPLICATION NOTES(back to Table of Content)General protocol notesSurface Areas Of Tissue Culture VesselsTubes Recommended For Use With Lipofectamine™ 2000Optimizing Plasmid DNA Transfections With Lipofectamine™ 2000Transfection ProtocolsCo-Transfection Of Sirna And Plasmid DNATransfection Of Fluorescently Labeled Oligos With Lipofectamine™ 2000Transfection Of Cells In 96-Well PlatesTransient Transfection Of Suspension CellsGeneral protocol notes(back to Table of Content)(back to Protocol and Application Notes)It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.DMEM or RPMI 1940 can be used instead of Opti-MEM when making Lipofectamine™ 2000 – DNA complexes.However, the efficiency of complex formation may not be as high as with Opti-MEM. Cells in PBS can be transfectedusing Lipofectamine™ 2000.As long as the cells are healthy in the PBS, the transfection is likely to work.For a general plasmid DNA transfection protocol, please refer to the product insert:/content/sfs/manuals/Lipofectamine™2000_man.pdfA general protocol for transfecting Stealth RNAi or siRNA into mammalian cells can be found at the following site:/content/sfs/manuals/stealth_sirna_tsf_lf2k_man.pdfSurface Areas of Tissue Culture Vessels(back to Table of Content)(back to Protocol and Application Notes)48-well 24-well 12-well 6-well 35-mm 60-mm 100-mm 150-mm T25 T75 Culture Vessel 96-well0.7 2 4 10 10 20 60 140 2575 Surface Area (cm2) 0.30.237.5plate0.4 1 2 5 5 10 30 70 12.5 Ratioto24-wellTubes recommended for use with Lipofectamine™ 2000(back to Table of Content)(back to Protocol and Application Notes)It is best to use polypropylene tubes when pre-mixing Lipofectamine 2000 ™ and DNA. Polystyrene may not work as well.Optimizing plasmid DNA transfections with Lipofectamine™ 2000(back to Table of Content)(back to Protocol and Application Notes)The conditions that could be optimized include Lipofectamine™ 2000 amount, DNA concentration, and cell number. Keeping two variables constant, vary the third.For example: to optimize the amount of Lipofectamine™ 2000 for transfection in a 24-well plate, start with cells at >90% confluency and use a fixed amount of DNA (0.8-1.2 µg). With cell number and DNA concentration held constant, vary the amount of Lipofectamine™ 2000 to determine the optimal concentration (usually 1.5-3 µl). In the same way, the cell number and amount of DNA can also be optimized.It is recommended to use a range of 0.5 to 5 µl of Lipofectamine™ 2000 per µg of DNA. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing eitherLipofectamine™ 2000 amount or DNA concentration. With careful optimization, this can be achieved with little impact on the level of transgene expression.Transfection efficiency is typically measured as the percentage of cells translating and accumulating the protein of interest for detection in the total population. If the levels of translation or protein accumulation are low, a lower transfection efficiency may be obtained. A transfection control such as our BLOCK-iT Fluorescent Oligo (catalog # 2013) is a more accuratemeasure of the efficiency of DNA delivery since its detection is independent of expression in the cell.The following citation discusses the effect of variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and media components (serum and antibiotics) on transfection with Lipofectamine™ 2000. In addition, it also looks at high throughput transfections, siRNA transfections, and transfection of primary neurons.Advanced transfection with Lipofectamine™ 2000 reagent: primary neurons, siRNA, and high-throughput applications - Methods, Volume 33, Issue 2, June 2004, Pages 95-103 Brian Dalby, Sharon Cates, Adam Harris, Elise C. Ohki, Mary L.Tilkins, Paul J. Price and Valentina C. CiccaroneThough most cells transfect well in the presence or absence of serum, there are a few such as HeLa cells and Normal Human Fibroblasts that give better transfection efficiency in the absence of serum.Cell lines successfully transfected with Lipofectamine™ 2000:293F293H,293 BE(2)C(w/oserum)CHO-K1 CHO-S (adherent) CHO-S (suspension in CD CHO media)COS-1 (w/o serum) COS7-L(w/o serum) Primary Human FibroblastsHT-29(w/oserum) HT-1080 MDCKMRC-5(w/oserum) PC12SK-BR3VeroCHOCHO-DG44MCF7MDA-MB-361HCT116H1299RKOHep3B,HepG2HeLa Rzneo HOSC3H/10T1/2NIH3T3JurkatK562HUVECS LoVoA549Some cell lines for which transfection protocols are available (at the cell lines database)(back to Table of Content)(back to Protocol and Application Notes)Cell type TransfectionEfficiency (%) Cells per well(24-well plate)Lipofectamine™ 2000µl per well in24-well plate293H99 2 x 105 2293F99 2 x 105 2BE(2)C77 2 x 105 2.5BHK21- 1.0 x 105 3.0CHO-K1- 1.2 x 105 2.5CHO-S(adherent) 96 1.5 x 105 2.5Cos 1- 8 x 104 3.0COS7L99 8 x 104 2.5CV-170 8 x 104 1.5HeLa94 8 x 104 1.5HT-29- 1.5 x 105 3.0HT108081 8 x 104 1.5HUVEC<2% 8.0 x 104 2.0MDCK43 6 x 104 4MRC-5Not measured 1.5 x 105 2.5Murine Embryonic Stem Cells, D3 (6-well plates)75 1X106 (6-well) 8-12(6-well)NIH3T3Not measured 1.5 x 105 2PC1285 2.5 X 105 2Primary Human Fibroblasts48 8 x 104 2Primary Human Keratinocytes- 8 x 104 2RKO (field test) (6-well plates) 40 - 60 See protocol See protocol SKBR349 1.5 x 105 2Vero86 8 x 104 2Rat Hepatocytes50 1.25 x 105 1.5Rat E18 Cortical Neurons20-25 2 x 105 4Co-transfection of siRNA and plasmid DNA(back to Table of Content)(back to Protocol and Application Notes)Plasmid and siRNA co-transfection are possible. Co-transfections have been tested with Lipofectamine™ 2000 in GripTite™ cells (293 derived cells) plated at 1.8 x 105 cells/well in a 24-well format (0.5ml medium, no antibiotics).200ng of two different reporter plasmids were co-transfected with 10pmol of siRNA following the standardLipofectamine™ 2000 protocol, with 2ul of Lipofectamine™ 2000 per well. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells.Transfection of fluorescently labeled oligos with Lipofectamine™ 2000(back to Table of Content)(back to Protocol and Application Notes)Fluorescently labeled oligos that depend on hairpin structures for quenching (like LUX primers) may fluoresce upon mixing with Lipofectamine™ 2000. In such cases Oligofectamine is suggested. Oligofectamine is very different chemically, and therefore not expected to exhibit the same strength of interaction.Transfection of cells in 96-well plates (also see Focus 21.3 page58)(back to Table of Content)(back to Protocol and Application Notes)Use the 24-well plate protocol with the following modifications:Plate 2-6 x 104 cells per well in 100 µl of the appropriate complete growth medium without antibiotics and with serum if cells are normally cultured in the presence of serum.For each well of cells, dilute 240 to 320 ng of DNA into 25 µl medium without serum (e.g., OptiMEM® I Medium) in 96-well, sterile micro titer plates.For each well of cells, dilute 0.8-1 µl of Lipofectamine™ 2000 into 25 µl OptiMEM® Medium and incubate for 5 min at room temperature. Once the Lipofectamine™ 2000 is diluted, combine it with the DNA within 30 min. Longerincubation times may result in decreased activity. This dilution can be prepared in bulk for multiple wells.Add 25 µl of the diluted Lipofectamine™ 2000 (from step 3) to each well containing diluted DNA (from step 2), mix gently, and incubate at room temperature for 20 min to allow DNA- Lipofectamine™ 2000 complexes to form.Add the DNA- Lipofectamine™ 2000 complexes (50 µl) directly to each well of the plates containing cells and mix gently.Optimal transfection conditions for transfections in 96-well plates:Cell Line Seeding Density(Cells per well) DNA per Well Lipofectamine™ 2000(µl)CHO-S 2 x 104240 ng 1 µlCOS-7L 2.5 x 104320 ng 1 µl293H, 293F 5 x 104320 ng 1 µlAlternate rapid protocol for 96-well transfections without pre-plating cells (also see Focus 21.3, page58): This protocol is designed as a rapid alternative that does not require plating cells the day before transfection. Instead, a suspension of cells is added directly to complexes prepared in 96-well plates. This protocol has been used successfully with the cells and conditions outlined below. Use poly-lysine coated plates (D or L) for best results.Dilute ~320 ng of each DNA to be tested into 25 µl medium without serum (e.g., OptiMEM® I Medium). Prepare the dilutions directly in 96-well cell culture plates.For each well, dilute 0.4-0.8 µl of Lipofectamine™ 2000 into 25 µl OptiMEM® I Medium and incubate for 5 min at room temperature. Prepare this dilution in bulk for multiple wells. Once the Lipofectamine™ 2000 is diluted, combine it with the DNA within 30 min. Longer incubation times may result in decreased activity.Add 25 µl of the diluted Lipofectamine™ 2000 (from step 2) to each well containing diluted DNA (from step 1), mix gently, and incubate at room temperature for 20 min to allow DNA-Lipofectamine™ 2000 complexes to form.Prepare a cell suspension so that the appropriate number of cells per well is contained in 100 µl of growth medium. Use approximately twice the cell density, depending on cell type, than with the standard protocol.Add 100 µl of the cell suspension (from step 4) to each of the wells containing the DNA-Lipofectamine™ 2000 complexes (from step 3) and mix gently.Incubate at 37o C in a CO2 incubator until ready to assay (24-48 h post transfection). It is not necessary to remove the complexes or change the medium. Cells will adhere as usual in the presence of the complexes.Transfection conditions for rapid 96-well protocol:Cell Line Cells per well DNA per well(100 µl suspension) Lipofectamine™ 2000 per wellCHO-S 5 x 104320 ng 0.8 µlCOS-7L 6 x 104320 ng 0.6-0.8 µl293H, 293F 1.2 x 105320 ng 0.4 µlTransient transfection of suspension cells(back to Table of Content)(back to Protocol and Application Notes)The following protocol was optimized with Jurkat and K562 cells, but can be used as a guideline for other types of suspension lines.For each transfection, add a cell suspension containing 4-8 x 105 cells in 500 µl of growth medium with serum but without antibiotics, to a well of a 24 well plate. For transfection of larger number of cells, scale up all the reagents (cells, media, DNA, Lipofectamine™ 2000 and plate size) proportionately to the number of cells transfected.For each well, dilute 0.8 - 1.2 µg of DNA into 50 µl of medium without serum (e.g., Opti-MEM). This can be prepared in bulk for multiple wells.For each well, dilute ~2 µl of Lipofectamine™ 2000 into 50 µl OptiMEM® I Medium and incubate for 5 min at room temperature. Once the Lipofectamine™ 2000 is diluted, combine it with the DNA within 30 min. Longer incubationtimes may result in decreased activity. This dilution can be prepared in bulk for multiple wells.Combine the diluted DNA from step 2 with the diluted Lipofectamine™ 2000 from step 3. Incubate at room temperature for 20 min to allow DNA-Lipofectamine™2000 complexes to form.Add the DNA-Lipofectamine™ 2000 complexes from step 4 (100 µl) directly to each well containing cells (from step 1) and mix gently by rocking the plate back and forth.Incubate for 4 h at 37o C in a CO2 incubator.In Jurkat cells, addition of PHA-L (Phytohemagglutinin L) and PMA (phorbol myristate acetate) at final concentrations of1 µg/ml and 50 ng/ml, respectively, enhances CMV promoter activity and gene expression. In K562 cells, PMA alone issufficient to enhance promoter activity. PMA and PHA are added after the 4-h incubation.Assay the cells at 24-48 h post-transfection for the appropriate activity. It is not necessary to remove the complexes or change the medium.Note: Jurkat cells are difficult to transfect and have low expression following transfection.The use of PHA-L and PMA did not affect the expression level of a beta-gal reporter (by ONPG assay) in either Jurkat cells orK562 cells in our hands. They are widely used in transfecting these cell types, and their use is most likely historical. Their ffectiveness in transfections with currently available lipid reagents has probably not been tested before.ePRODUCT DOCUMENTATION(back to Table of Content)Brochures Cell Lines CitationsCOA FAQ LicensingManuals MSDSREFERENCES(back to Table of Content)Krista Evans, et al..PGreen Lantern-1, A Superior Green Fluorescent Protein Mammalian Cell Transfection Reporter, Focus 18(2): 40.Valentina Ciccarone, et al. Lipofectamine™ 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells - Focus 21(2):54.Jean-Pierre Pichet and Valentina Ciccarone. Transfection of Mammalian Cells in 96-Well Plates with Lipofectamine™ 2000 Reagent. Focus 21(3):58.Cationic Lipid Reagent Selection. Focus 21(3):61.Linda Roy, et al. High Transfection Efficiency of Cloned Cell Lines. Focus 21(3):62.Achieve the highest transfection efficiencies and higher expression levels (with Lipofectamine™ 2000). Expressions 8(3):18.PRODUCT NAME AND CATALOG NUMBERS(back to Table of Content)Name Size Part Number Catalog Nnumber Lipofectamine™ 2000 Reagent 0.75 ml 52758 11668-027(11668027) Lipofectamine™ 2000 Reagent 1.5 ml 52887 11668-019(11668019) Lipofectamine™ 2000 CD Reagent 1.0 ml 52888 12566-014(12566014) ASSOCIATED PRODUCTS(back to Table of Content)OPTI-MEM I Reduced Serum Medium (catalog # 31985-062)AntibioticsGIBCO® Cell Culture ProductsNeed more help? Please email us by clicking here.。
lipofectamine 2000
细胞转染
(1) 转染前一天，用胰酶消化对数生长期的BGC细胞并计数，以3.5×105/孔，将细胞接种于2mL 含10%胎牛血清的无抗生素培养基的6孔培养板中。
(2) 铺板次日，待细胞贴壁生长汇合度约80%，将6 孔板中每个孔的旧培养基吸尽，用不含血清的OPTI-MEM 培养基洗涤细胞2 次，然后 6 孔板每个孔中加入1.5mL 的无血清的OPTI-MEM 培养基。
(3) 取出Trop-2载体，空载体和脂质体Lipofectamine 2000 放置室温中，使其融化。
(4) 对于每孔细胞，用200ul OPTI-MEM 培养基分别稀释Trop-2载体2微克，空载体1微克，轻轻混合。
(5)取5u1 Lipofectamine 2000 缓慢加入至200ul OPTI-MEM 培养基中，并将两者轻轻混均，室温孵育5 分钟。
(25min内进行下一步操作)
(6) 将稀释后的Lipofectamine 2000 分别与Trop-2载体，空载体混合，轻柔操作，以防破坏Lipofectamine 2000 和RNA 链断裂，室温孵育20 分钟。
(7) 缓慢均匀的将上述复合物加入到相应孔中，摇动培养板，轻轻混匀。
(8) 将培养板放入37℃，5%的CO2 培养箱中孵育6 小时后，换含10%胎牛血清的无抗生素1640 培养液继续。
lip2000转染说明书
4. 哺乳动物细胞siRNA转染4.1 转染方法：将制备好的siRNA、siRNA表达载体或表达框架转导至真核细胞中的方法主要有以下几种：磷酸钙共沉淀；电穿孔法；DEAE-葡聚糖和polybrene；机械法；阳离子脂质体试剂。
目前用的最多的是阳离子脂质体法。
4.1.1 磷酸钙共沉淀将氯化钙，RNA(或DNA)和磷酸缓冲液混合，沉淀形成包含DNA且极小的不溶的磷酸钙颗粒。
磷酸钙-DNA复合物粘附到细胞膜并通过胞饮进入目的细胞的细胞质。
沉淀物的大小和质量对于磷酸钙转染的成功至关重要。
在实验中使用的每种试剂都必须小心校准，保证质量，因为甚至偏离最优条件十分之一个pH都会导致磷酸钙转染的失败。
4.1.2 电穿孔法电穿孔通过将细胞暴露在短暂的高场强电脉冲中转导分子。
将细胞悬浮液置于电场中会诱导沿细胞膜的电压差异，据认为这种电压差异会导致细胞膜暂时穿孔。
电脉冲和场强的优化对于成功的转染非常重要，因为过高的场强和过长的电脉冲时间会不可逆地伤害细胞膜而裂解细胞。
一般，成功的电穿孔过程都伴随高水平（50%或更高）的毒性。
4.1.3 DEAE-葡聚糖和polybrene带正电的DEAE-葡聚糖或polybrene多聚体复合物和带负电的DNA分子使得DNA 可以结合在细胞表面。
通过使用DMSO或甘油获得的渗透休克将DNA复合体导入。
两种试剂都已成功用于转染。
DEAE-葡聚糖仅限于瞬时转染。
4.1.4 机械法转染技术也包括使用机械的方法，比如显微注射和基因枪（biolistic particle）。
显微注射使用一根细针头将DNA，RNA或蛋白直接转入细胞质或细胞核。
基因枪使用高压microprojectile将大分子导入细胞。
4.1.5 阳离子脂质体试剂在优化条件下将阳离子脂质体试剂加入水中时，其可以形成微小的（平均大小约100-400nm）单层脂质体。
这些脂质体带正电，可以靠静电作用结合到DNA的磷酸骨架上以及带负电的细胞膜表面。
Lipofectamine 2000细胞转染实验步骤 注意事项
Invitrogen阳离子转染试剂Lipofectamine 2000细胞转染实验步骤注意事项2010-07-10 16:16Invitrogen的细胞转染试剂：Lipofectamine 2000Lipofectamine 2000是最为人熟知的转染产品之一。
已知可为517种细胞（见下面连接地址）提供高转染效率（表达转基因细胞的百分数）和活性（细胞抽提物中转入基因的酶产物活性）。
特点两个关键性特点使得Lipofectamine 2000试剂的转染步骤快速简便：（1）DNA-阳离子脂质体试剂的复合体可以直接加入到细胞培养基中，有血清也不怕（2）转染后不需要除去Lipofectamine 2000试剂，无需换培养基操作流程事实上Lipofectamine系列产品操作流程都是又快又简单：稀释DNA 以及Lipofectamine 2000，混合2种稀释液保温20分钟，加入培养细胞中孵育24－96小时检测结果。
下面是Invitrogen提供的详细流程和注意事项。
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%。
细胞铺板在0.5ml含血清，不含抗生素的正常生长的培养基中。
对于每孔细胞，使用50μl无血清培养基（如OPTI-MEMⅠ培养基）稀释0.8μg-1.0μg DNA。
对于每孔细胞，使用50μl OPTI-MEMⅠ培养基稀释1μl-3μl LIPOFECTAMINE 2000试剂。
Lipofectamine 2000稀释后保温5分钟（在30分钟内同稀释的DNA 混合。
保温时间过长会降低活性。
）注意：即使Lipofectamine 2000使用OPTI-MEMⅠ稀释，细胞也可以使用D-MEM培养。
如果D-MEM做为Lipofectamine 2000的稀释液，必须在5分钟内同稀释的DNA混合。
混合稀释的DNA（第2步）和稀释的Lipofectamine 2000（第3步）。
在室温保温20分钟。
lipo2000转染操作步骤
Lipo2000 瞬时转染细胞步骤Stealth™ RNAi or siRNA Transfection以24孔板为例，其余规格的转染见表11 中板，细胞密度为30-50%适宜。
注意：根据转染后细胞检测时间长短决定细胞中板密度，如果转染后需要长时间后检测，则细胞中板密度适当降低，已避免细胞过度生长导致存活降低。
2 第二天（24-36小时后）每个孔转染方式如下：A 将20pmol siRNA溶于50ul Opti-mem无血清培养基中。
B 将1ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。
C 将A B两管混合，放置20min。
3 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。
将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。
Plasmid DNA TransfectionDNA(ug):lipo 2000(ul)=1:2-3转染时细胞密度越高，转染效率，表达效率也越高，并且可以降低细胞毒性。
1 中板。
贴壁细胞：0.5-2X105 cells/well，第二天待细胞密度达到70-80%时转染悬浮细胞：4-8X105 cells/well，中板后随即转染。
2 转染。
A 将0.8ug DNA溶于50ul Opti-mem无血清培养基中。
B 将2ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min。
C 将A B两管混合，放置20min。
转染期间，将24孔板培养基换成无血清培养基，每孔400ul。
将C管mix加入24孔板对应孔中，4-6小时候换成有血清培养基。
Table 1. Culture Shared reagents DNA transfection RNAi transfection*：中板密度根据不同细胞不同实验有所不同，这里仅提的数据仅供参考**：6孔板细胞质粒转染量1-2ug足以。
***：6cm dish细胞质粒转染量4-6ug足以。
lipo 2000说明书
Lipofectamine™2000转染说明书IntroductionLipofectamine™ 2000 CD is a proprietary animal origin-free formulation for the transfection of nucleic acids into eukaryotic cells. A Drug Master File (DMF) has been submitted to the FDA. Contact Technical Service or your local Sales Representative for permission to cross-reference the DMF. Using Lipofectamine™ 2000 CD for transfection provides the following advantages:Highest transfection efficiency in many cell types and formats. Refer to the Cell Lines database at for a list of cell types transfected. he animalorigin-free formulation ensures that mammalian cell culture and bioproduction processes are free of animal-derived materials. DNA-Lipofectamine™ 2000 CD complexes can be added directly to cells in culture medium. It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.Important Guidelines for Transfection1.Culture cells in serum-free media that are free of animal-derived components.Test serum-free media for compatibility with Lipofec tamine™ 2000 CD since some serum-free formulations (e.g. CD 293, 293 SFM II, CD Hybridoma)may inhibit cationic lipid-mediated transfection.2.For consistent animal origin-free transfection, use OptiPro™ SFM (Cat. No.12309-019) to dilute DNA and Lipofecta mine™ 2000 CD before complexing.3.Transfect cells at high cell density: For adherent cells, 90-95% confluence atthe time of transfection is recommended for high efficiency, high expressionlevels, and to minimize cytotoxicity. For suspension cultures, transfect cells ata density of 0.8-1.1 x 106 cells/ml. Optimization may be necessary. Maintainthe same seeding conditions between experiments.4.Do not add antibiotics to media during transfection as this causes cell death.5.Do not add Pluronic® or charged media extracts (e.g. dextran sulfate) tomedia during transfection as these reagents can inhibit transfection Transfecting Adherent CellsUse the following procedure to transfect mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. Use the recommended Lipofectamine™ 2000 CD amount as a starting point, then optimize conditions for your cell line and serum-free medium, as needed. All amounts and volumes are given on a per well basis.1.One day before transfection, plate 0.5-2 x 105 cells in 500 μl of growthmedium without antibiotics so that they will be 90-95% confluent at the timeof transfection.2.For each transfection sample, prepare complexes as followso a. Dilute DNA in 50 μl of OptiPro™ SFM. Mix gen tly.o b. Mix Lipofectamine™ 2000 CD gently before use, then dilute the appropriate amount in 50 μl of OptiPro™ SFM. Incubate for 5 minutesat room temperature. Note: Combine diluted Lipofectamine™ 2000CD with diluted DNA within 30 minutes.o c. After 5 minute incubation, combine the diluted DNA with diluted Lipofectamine™ 2000 CD (total volume = 100 μl). Mix gently andincubate for 20 minutes at room temperature (solution may appearcloudy). Note: Complexes are stable for 6 hours at room temperature.3.Add the 100 μl of complexes to a well containing cells and medium. Mixgently by rocking the plate back and forth.4.Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing fortransgene expression. It is not necessary to change the medium, but mediummay be replaced after 4-6 hours.5.For stable cell lines: Passage cells at a 1:10 (or higher dilution) into freshgrowth medium 24 hours after transfection. Add selective medium thefollowing day.TOP Scaling Up or Down TransfectionsTo transfect cells in different tissue culture formats, vary the amounts of Lipofectamine™ 2000 CD, DNA, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 μl is recommended for transfections in 96- well plates.Culture vessel Surf. areaper well(cm2)Relativesurf. areavs. 24-wellVol. ofplatingmediumDNA (μg) inmedia vol. (μl)Lipofectamine™2000 CD (μl) inmedia vol. (μl)96-well 0.3 0.2 100 μl0.2 μg in 25 μl0.5 μl in 25 μl 24-well 2 1 500 μl0.8 μg in 50 μl 2.0 μl in 50 μl12-well 4 2 1 ml 1.6 μg in 100μl4.0 μl in 100 μl35-mm 10 5 4.0 μg in 250μl10 μl in 250 μl6-well 10 5 2 ml 4.0 μg in 250μl10 μl in 250 μl60-mm 20 10 5 ml 8.0 μg in 0.5ml20 μl in 0.5 ml10-cm 60 30 15 ml 24 μg in 1.5 ml 60 μl in 1.5 mlNote: Surface areas are determined from actual measurements of tissue culture vessels, and may vary depending on the manufacturer.Transfecting Cells in SuspensionUse the following procedure to transfect suspension cells at any scale. Before beginning, make sure that cells are healthy and > 90% viable.1.On the day of transfection, prepare a single cell suspension from stock cells at< 3 x 106 cells/ml. Seed cells at a density of 0.8-1.1 x 106 cells/ml in growthmedia without antibiotics.2.For each transfection sample, prepare complexes as in Step 2, using thefollowing reagent amounts and volumes for every ml of cells transfected:o Dilute 0.5-1.5 μg DNA in 34 μl of OptiPro™ SFMo Dilute 1-10 μl of Lipofectamine™ 2000 CD in 34 μl of OptiPro™ SFM3.Add the complexes to the flask containing cells and media.4.Incubate the cells at 37°C in a CO2 incubator on an orbital shaker rotating at125 rpm for 24-96 hours prior to testing for transgene expression.Optimizing TransfectionTo obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying cell density and Lipofectamine™ 2000 CD concentrations.Quality ControlLipofectamine™ 2000 CD is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.。
LIPOFECTAMINE2000转染试剂转染步骤
LIPOFECTAMINE2000转染试剂转染步骤1.准备转染试剂：取出存储在-20℃的LIPOFECTAMINE2000试剂，并将其溶解在适量的去离子水或者PBS缓冲液中，制备转染试剂。
2. 根据实验需要确定转染的质粒DNA量和细胞数量。
一般来说，每个转染需要1-2ug的质粒DNA，细胞密度则根据细胞类型的不同而有所变化。
3.将准备好的转染试剂和质粒DNA混合在一起。
首先将质粒DNA加入到含有LIPOFECTAMINE2000的管中，并轻轻混合均匀，然后将混合物静置15-30分钟，使其形成脂质-DNA复合物。
4.在脂质-DNA复合物静置的同时，准备待转染的细胞。
将细胞用无血清培养基洗涤一次，并将其悬浮在新的无血清培养基中。
5.将静置好的脂质-DNA复合物滴加到细胞中。
将脂质-DNA复合物滴加到含有细胞的培养皿中，并轻轻摇晃培养皿，使复合物均匀分布在细胞表面。
6.将转染后的细胞培养在37℃的CO2培养箱中孵育。
具体培养时间视实验需求而定，一般来说，24-48小时后可以进行下一步实验。
7. 检测转染效率。
可以通过荧光显微镜观察细胞内是否表达了目的基因或荧光标记，也可以采用Western blotting或者RT-PCR等方法进行进一步的检测。
总的来说，LIPOFECTAMINE2000转染试剂转染步骤相对简单，但需要注意的是在每一步操作中都要轻柔并避免产生气泡，以确保脂质-DNA复合物可以均匀地与细胞相结合，从而提高转染效率。
同时，在实验过程中需要注意质粒DNA的质量和浓度，以及细胞的健康状态，这些因素都会对转染效果产生影响。
希望以上介绍对您有所帮助，祝您实验顺利。
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L i p o f e c t a m i n e
2000转染说明-CAL-FENGHAI.-(YICAI)-Company One1
Lipofectamine TM 2000
CAT. NO. 11668-027 Size:
CAT. NO. 11668-019 Size: ml
4℃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%。
悬浮细胞：在准备转染混合物时，每500μl无抗生素培养基中含有细胞数量为4-8×105。
2.对于每个转染样品，按下面的方法准备：
a、在50μl无血清Opti-MEM I 低血清培养基中（或是其它无血清培养基）
稀释DNA，并轻轻混匀。
b、使用前轻轻混匀Lipofectamine TM 2000，然后取相应的量稀释在
Opti-MEM 培养基中。
室温下静置5分钟。
注意：要在30分钟内混合
Lipofectamine TM 2000和DNA的稀释液。
c、室温下静置5分钟后，混合Lipofectamine TM 2000和DNA的稀释液（总
体积为100μl）。
轻轻混匀并在室温下静置20分钟（溶液混合后可能会看上去浑浊）。
注意：混合物在室温下的6个小时内不会失效。
3.细胞板的每个孔中加混合液100μl，并前后轻轻摇动细胞板使混合液与孔中
的培养液混匀。
4.检测目的基因的表达情况前，细胞于37℃ CO2培养箱中培养18-48小时。
此时不需要改变培养基，但4-6小时后也许需要更换.
5.对于固定的细胞系：转染24小时后将细胞以1：10（或更高的稀释度）稀
释度转移到新鲜的生长培养基中。
如果需要的话，在以后的培养中可以在培养基中加入选择培养基。
对于悬浮的细胞系：转染4小时后，如果需要，加入PMA和/或PHA以提高CVM启动子的活性并促进基因表达。
转染量度标准：
在不同的组织培养板中转染细胞时，根据相对的表面积，按比例加入Lipofectamine TM 2000、细胞、和培养基，具体情况剪下表。
在自动、高产率体系中，建议96孔板的转染混合物的体积为50μl。
注意:操作时，可以直接将细胞混入转染混合物中进行快速的96孔板转染。
在细胞板中准备好转染混合物，然后直接加入说明中正常100μl体积时浓度的2倍浓度细胞培养液。
在转染混合液存在情况下，细胞可以正常的贴壁。
发生变化。
优化转染：
为了得到高的转染效率和低的无用副效应，通过改变细胞浓度、DNA以及Lipofectamine TM 2000的浓度来优化转染条件。
另外，要保证细胞培养基的细胞混浊度高于90%，DNA(μg)与Lipofectamine TM（μl ）2000的比例在1：到1：5之间。