Lipofectamine 2000 Transfection Reagent

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原理Lipofectamine2000是一种常用于转染细胞的试剂，它的原理是通过脂质体介导的转染技术，将外源DNA或RNA导入到细胞内。

这种转染方法被广泛应用于基因功能研究、蛋白表达和基因治疗等领域。

脂质体是由脂质分子组成的小囊泡，可以与细胞膜融合并释放其内部所带的DNA或RNA分子。

Lipofectamine2000是由一种阳离子脂质和一种阴离子脂质组成的复合物，这种复合物能够与DNA 或RNA形成稳定的复合体。

当Lipofectamine2000和DNA或RNA共同存在于培养基中时，它们会自发地结合在一起，形成脂质体-核酸复合物。

脂质体-核酸复合物具有良好的转染效果，主要有以下几个原因。

首先，脂质体可以提供稳定的保护作用，保护DNA或RNA免受外界环境的影响。

其次，脂质体能够与细胞膜融合并被细胞摄取，将DNA或RNA导入到细胞内。

此外，Lipofectamine2000还可以增加细胞膜的通透性，使DNA或RNA更容易进入细胞。

Lipofectamine2000的使用方法相对简单。

首先，将所需的DNA 或RNA与Lipofectamine2000按照一定比例混合，形成脂质体-核酸复合物。

然后将复合物加入到细胞培养基中，与细胞共同孵育一段时间。

在此过程中，脂质体-核酸复合物与细胞相互作用，将DNA或RNA导入到细胞内。

最后，可以利用适当的实验方法检测转染效果，如荧光显微镜观察、PCR检测等。

尽管Lipofectamine2000在转染实验中具有诸多优势，但也存在一些局限性。

首先，该方法对细胞类型有一定的选择性，不同细胞株对Lipofectamine2000的响应程度不同。

其次，脂质体-核酸复合物的稳定性较差，容易受到环境因素的影响。

此外，Lipofectamine2000的转染效率可能受到多种因素的影响，如DNA或RNA浓度、孵育时间等。

为了提高转染效率，研究人员还不断改进Lipofectamine2000的配方和使用方法。

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。

Lipofectamine™ 2000Cat. No. 11668-027 Size: 0.75 mlCat. No. 11668-019 Size: 1.5 mlCat. No. 11668-500 Size: 15 mlStore at +4°C (do not freeze) DescriptionLipofectamine™ 2000 is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells providing the following advantages:•Highest transfection efficiency in many cell types and formats (e.g. 96-well).Refer to the Cell Lines database at for a list of cell types successfully transfected.•Nucleic acid-Lipofectamine™ 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.•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 Transfection•Use the procedure on page 2 to transfect cells with short interfering RNA (siRNA) or Stealth™ RNAi.•Use the procedure on page 3 to transfect cells with plasmid DNA.•We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine™ 2000 and nucleic acids before complexing.•Do not add antibiotics to media during transfection as this causes cell death. •Maintain the same seeding conditions between experiments.•Test serum-free media for compatibility with Lipofectamine™ 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibitcationic lipid-mediated transfection.Note:For more tips for your RNAi experiment, refer to “Seven Steps to RNAi Success”. This manual is available from /rnai or Technical Service, as are cell-type specific RNAi transfection protocols (see “RNAi protocols”.)Quality ControlLipofectamine™2000 is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.Part No.: 11668.2k.pps Rev. Date: 11 July 2006For research use only. Not intended for any animal or human therapeutic or diagnostic use.For technical support, contact tech_service@.Page 2 Stealth™ RNAi or siRNA TransfectionUse this brief procedure to transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections (page 4). All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Stealth™ RNAi or siRNA Transfection, especially if you are transfecting a mammalian cell line for the first time.1.One day before transfection, plate cells in 500 µl of growth medium withoutantibiotics such that they will be 30-50% confluent at the time of transfection.Note: Transfecting cells at a lower density allows a longer interval between transfection and assay time, and minimizes the loss of cell viability due to cell overgrowth.2.For each transfection sample, prepare oligomer-Lipofectamine™ 2000complexes as follows:a.Dilute 20 pmol Stealth™ RNAi or siRNA oligomer in 50 µl Opti-MEM® IReduced Serum Medium without serum (final concentration of RNA when added to the cells is 33 nM). Mix gently.b.Mix Lipofectamine™ 2000 gently before use, then dilute 1 µl in 50 µl Opti-MEM® I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.c.After the 5-minute incubation, combine the diluted oligomer with thediluted Lipofectamine™ 2000. Mix gently and incubate for 20 minutes atroom temperature (solution may appear cloudy).3.Add the oligomer-Lipofectamine™ 2000 complexes to each well containingcells and medium. Mix gently by rocking the plate back and forth.Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours. Optimizing Stealth™ RNAi or siRNA TransfectionTo obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine™ 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 µl Lipofectamine™ 2000 for 24-well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.Page 3 Plasmid DNA TransfectionUse the following procedure to transfect DNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections (page 4). All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (µg) to Lipofectamine™ 2000 (µl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. Optimization may be necessary (see Optimizing Plasmid DNA Transfection, page 4).1.Adherent cells: One day before transfection, plate 0.5-2 x 105cells in 500 µl ofgrowth medium without antibiotics so that cells will be 90-95% confluent at the time of transfection.Suspension cells: Just prior to preparing complexes, plate 4-8 x 105 cells in 500 µl of growth medium without antibiotics.2.For each transfection sample, prepare complexes as follows:a.Dilute DNA in 50 µl of Opti-MEM® I Reduced Serum Medium withoutserum (or other medium without serum). Mix gently.b.Mix Lipofectamine™ 2000 gently before use, then dilute the appropriateamount in 50 µl of Opti-MEM® I Medium. Incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.c.After the 5 minute incubation, combine the diluted DNA with dilutedLipofectamine™ 2000 (total volume = 100 µl). Mix gently and incubate for20 minutes at room temperature (solution may appear cloudy). Note:Complexes are stable for 6 hours at room temperature.3.Add the 100 µl of complexes to each 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. Medium may be changed 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 (ifdesired) the following day.Page 4Optimizing Plasmid DNA TransfectionTo obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as DNA andLipofectamine ™ 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA (µg): Lipofectamine ™ 2000 (µl) ratios from 1:0.5 to 1:5. Scaling Up or Down TransfectionsTo transfect cells in different tissue culture formats, vary the amounts of Lipofectamine ™ 2000, nucleic acid, 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. Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume. Cells will adhere as usual in the presence of complexes.Shared reagents DNA transfection RNAi transfection Culturevessel Surf. area perwell 1 Vol. of plating medium Vol. of dilution medium 2 DNA Lipofect-amine ™ 2000RNA Lipofect-amine ™2000 96-well 0.3 cm 2 100 µl 2 x 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl24-well 2 cm 2 500 µl2 x 50 µl 0.8 µg 2.0 µl 20 pmol 1.0 µl 12-well 4 cm 2 1 ml2 x 100 µl 1.6 µg 4.0 µl 40 pmol 2.0 µl 6-well 10 cm 2 2 ml2 x 250 µl 4.0 µg 10 µl 100 pmol 5 µl 60-mm 20 cm 2 5 ml2 x 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl 10-cm 60 cm 215 ml 2 x 1.5 ml 24 µg 60 µl 600 pmol 30 µl 1Surface areas may vary depending on the manufacturer. 2 Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols. Purchaser NotificationThis product is covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or our web-site, ). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.Limited Use Label License No. 27: Lipofectamine ™ 2000 ReagentLimited Use Label License No. 173: Inhibition of Gene Expression by Double-Stranded RNA Limited Use Label License No. 196: Stealth ™ RNAi©2000-2006 Invitrogen Corporation. All rights reserved.。

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。

LIPOFECTAMINE2000转染试剂转染步骤转染是一种将外源DNA或RNA导入到细胞内的技术，以研究基因功能、蛋白质表达、细胞信号转导等方面的问题。

LIPOFECTAMINE2000是一种常用的转染试剂，广泛应用于多种细胞系中。

以下是LIPOFECTAMINE2000转染试剂转染步骤的详细介绍。

一、细胞种植与处理准备1.1细胞传代：将细胞进行传代，以保证其在良好的状态下进行实验。

1.2细胞密度调整：将细胞于适宜培养皿中培养至60-80%的密度，以保证细胞的适宜转染。

1.3细胞处理准备：在转染前，将细胞用无酶EDTA或胰酶剥离并重新悬浮在适宜的培养基中，以保持细胞的完整性和适宜的状态。

二、试剂配制2.1DNA或RNA的制备：将外源DNA或RNA在无菌条件下制备，并使用纯化试剂进行纯化和浓缩。

2.2转染试剂配制：将冻干的LIPOFECTAMINE2000转染试剂通过加入合适的无菌水，稀释成适宜浓度的转染试剂。

三、转染操作3.1转染试剂与DNA/RNA的混合：将适量的LIPOFECTAMINE2000转染试剂与DNA或RNA混合在无菌的管中，轻轻混合均匀。

注意，避免过量试剂和核酸的使用，以减少细胞的毒性和副作用。

3.2孵育混合物：将混合物在常温条件下孵育15-30分钟，以促使脂质体与核酸形成稳定的复合体。

四、转染过程4.1转染试剂与细胞的混合：将混合物缓慢滴加到处理好的细胞培养基上，缓慢摇晃培养皿以使混合物均匀分布。

4.2转染时间及培养条件：将细胞放置在转染液中，保持静止状态，同时将培养皿放回培养箱中，在37℃、5%CO2的恒温恒湿条件下进行转染。

转染时间需要根据细胞系和转染试剂的要求进行优化，一般为4-6小时。

4.3转染液的去除：将转染液小心去除，并将细胞用含有适宜抗生素或筛选剂的培养基洗涤一次，以去除残留的转染试剂。

五、细胞处理及分析5.1细胞培养：将细胞放回恒温恒湿培养箱中，用适宜培养基进行细胞的培养。

赛默飞lipo2000说明书产品概述：值得信赖的、简单的广谱转染试剂，适用于大多数细胞系。

转染；通常是指将核酸引入真核细胞，或者更具体地说，引入动物细胞中。

Invitrogen Lipofectamine 2000 转染试剂是一种多功能转染试剂，经证明可以将各种有效载荷有效地转染到各种贴壁和悬浮细胞系中。

研究人员将 Lipofectamine 2000 试剂用于质粒 DNA 转染以及基于 siRNA 和 shRNA 的基因敲除实验、基因表达研究。

使用 Lipofectamine 2000 试剂，您可实现：适用各种细胞系，转染效率极高，重组蛋白表达水平高，实验方案简单siRNA 和质粒 DNA 共转染性能出众、可靠，适合各种高通量应用Lipofectamine 2000 转染试剂的使用被更多出版物引用，远超其他转染试剂进行可靠的细胞转染：图 1.Lipofectamine 2000 试剂使用方案步骤概要。

Lipofectamine 2000 试剂以其出色的转染性能为蛋白表达、基因沉默和功能测定递送DNA 或 siRNA。

这种广谱试剂效率超高，实验方案简单，成功应用于各种细胞系，因此倍受欢迎（图 1）。

查看下列实验方案。

对于基因沉默，高效转染提供高水平的基因敲除，从而获得令人信服的结果Lipofectamine 2000 转染试剂能够有效转染 siRNA 和质粒 DNA，因此是共转染的极佳选择能够为自动化或机器人系统轻松创造转染条件，因而非常适合高通量工作高性能常见转染试剂：Lipofectamine 2000 转染试剂可有效处理所有常见细胞系（图 2）和多种难以转染的细胞系，可在含或不含血清的培养基中使用。

查看下面“相关资源”部分中的实验方案。

图 2.使用 Lipofectamine 2000 试剂进行转染后的高水平 GFP 表达。

Lipofectamine 试剂的认可度：Lipofectamine 2000 试剂于 1999 年上市，此后引用该试剂的出版物数量稳步上升，该产品仍然是被引用次数最多的在售转染试剂之一（图 3）。

.Lipo2000 瞬时转染细胞步骤RNAi or siRNA Transfection Stealth?1 24孔板为例，其余规格的转染见表以适宜30-50%。

1 中板，细胞密度为如果转染后需要长时间注意：根据转染后细胞检测时间长短决定细胞中板密度，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。

24-36小时后）每个孔转染方式如下：2 第二天（无血清培养基中。

溶于A 将20pmol siRNA50ul Opti-mem 。

B 将1ul lipo2000溶于50ul Opti-mem无血清培养基中，混匀室温放置5min 两管混合，放置C 将A B20min。

加管mix3 转染期间，将24孔板培养基换成无血清培养基，每孔400ul。

将C 孔板对应孔中，4-6小时候换成有血清培养基。

入24Plasmid 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。

70-80%5 时转染 cells/well贴壁细胞：0.5-2X10，第二天待细胞密度达到 5 ，中板后随即转染。

4-8X10cells/well悬浮细胞：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。

1 / 3.：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* 足以。

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***：和碳酸氢钠进行缓HEPES 其中使用了减血清培养基是EMEM 的改良型,Opti-MEM? I常用其作为无.谷氨酰胺、痕量元素和生长因子并添加次黄嘌呤、胸苷、丙酮酸钠、冲,L-lip2000分别混合。

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.。

Reverse Transfection of Stealth™ RNAi or siRNA using Lipofectamine™ 2000 in a 96 well plate formatIntroductionLipofectamine™ 2000 Reagent is a proprietary formulation that facilitates highly efficient delivery of Stealth™ RNA moleculesor short interfering RNA (siRNA) to mammalian cells for RNAi analysis (1, 2). Here, we describe reverse transfection using Lipofectamine™ 2000. Reverse transfection is a method for high throughput (HTP) transfection of multiple siRNA or Stealth™RNAi duplexes, and is well suited in combination with RNAi duplexes pre-dispensed in 96-wells plates, such as the Stealth™RNAi Human Collections (available through our website, ).Reverse transfection combines RNA, transfection reagent, and cells in an altered sequence compared to traditional Lipofectamine™ 2000 transfection protocols. In short, a different RNAi molecule is put in each well prior to transfection and combined with diluted Lipofectamine™ 2000 to form complexes in each well. Cells are added directly to the Lipofectamine™2000-RNA complexes and transfection occurs while cells are attaching to the well.General Guidelines for TransfectionFollow these general guidelines when using Lipofectamine™ 2000 to reverse transfect siRNA or Stealth™ RNAi duplexes into mammalian cells.• Use low-passage cells, and make sure that cells are healthy and greater than 90% viable before transfection. This protocol is for use with adherent cells, as transfection efficiency for suspension cells generally is lower and may need optimization. • Do not add antibiotics to the medium during transfection as this reduces transfection efficiency and causes cell death.For optimal results, use Opti-MEM® I Reduced Serum Medium to dilute Lipofectamine™ 2000, DNA, and dsRNA oligomers prior to complex formation.• To increase accuracy and reduce assay variability, we recommend performing triplicate transfections for each sample condition.Materials NeededYou should have the following materials on hand before beginning:• Mammalian cell line cultured in the appropriate growth medium• Stealth™ RNAi or siRNA of interest (20 µM stock in 1X RNA Annealing/Dilution Buffer)• RNAi controls• Lipofectamine™ 2000 Reagent (Catalog nos. 11668-027 or 11668-019; store at +4°C and mix gently before use)• Opti-MEM® I Reduced Serum Medium (pre-warmed; Catalog nos. 31985-062 or 31985-070)• 96-well tissue culture plates and tissue culture suppliesTransfection ProcedureUse this procedure to reverse transfect your siRNA or Stealth™ RNAi duplexes into mammalian cells in 96-wells plates using Lipofectamine™ 2000. The complexes are prepared inside the wells, after which cells and medium are added. Remember to include the proper positive and negative controls in your experiment.1. For each well to be transfected, prepare DNA-RNAi molecule-Lipofectamine™ 2000 complexes as follows.a. Mix Lipofectamine™ 2000 gently before use, then dilute 0.25 µl Lipofectamine™ 2000 in 25 µl Opti-MEM® I Mediumwithout serum in a separate vessel. Mix gently and incubate for 5 minutes at room temperature.b. Dilute 15 pmol siRNA or Stealth™ RNAi in 25 µl Opti-MEM® I Medium without serum in the well of the tissue cultureplate. Mix gently.c. After the 5 minute incubation, add the diluted Lipofectamine™ 2000 to the wells with the diluted RNAi molecules. Mixgently and incubate for 15 minutes at room temperature to allow complex formation to occur. The solution may appear cloudy, but this will not impede the transfection.2. Add 100 µl complete growth medium without antibiotics with 20,000 cells to each well containing RNAi molecule-Lipofectamine™ 2000 complexes. This gives a final volume of 150 µl and a final RNA concentration of 100 nM. Mix gently by rocking the plate back and forth.3. Incubate the cells at 37°C in a CO2 incubator until you are ready to harvest cells and assay for your target gene.We recommend harvesting cells 24-48 hours after transfection to assay for target gene knockdown.Part no. 250877w.pps Rev. Date: 8 Sep 2005 For research use only. Not intended for any animal or human therapeutic or diagnostic use.For technical support, contact tech_service@.Page 2 Transfection Optimization.• 0.25 µl Lipofectamine™ 2000 per well is recommended as a starting point but optimization of transfection conditions may be required. A range of 0.125 µl to 1 µl is recommended.• Note that for highly potent RNAi molecules (i.e. RNAi molecules inducing > 90% target knockdown), the amount of dsRNA required to obtain effective knockdown may be less than the amounts specified. This needs to be determined empirically for each cell line.Limited Use Label License No. 27: Lipofectamine™ 2000The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Use of this product in conjunction with methods for the introduction of RNA molecules into cells may require licenses to one or more patents or patent applications. Users of these products should determine if any licenses are required. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.Limited Use Label License No. 173: Inhibition of Gene Expression by Double-Stranded RNAThis product’s and/or its use may be covered by one or more of U.S. Patent No. 6,506,559 and/or foreign equivalents, and is sold under license to Invitrogen Corporation by the Carnegie Institution of Washington, 1530 P Street, N.W. Washington, DC 20005. A separate license from the Carnegie Institution of Washington may be required to use this product.Limited Use Label License No. 196: Stealth™ RNAiThe purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.References1. Gitlin, L., Karelsky, S., and Andino, R. (2002) Nature 418, 430-434.2. Yu, J.Y., DeRuiter, S.L., and Turner, D.L. (2002) Proc. Natl. Acad. Sci. USA 99, 6047-6052.©2005 Invitrogen Corporation. All rights reserved.。

Lipofect 脂质体转染试剂简介：外源基因导入真核细胞的方法有很多种，如磷酸钙转染法、DEAE-葡聚糖转染法、脂质体法、电穿孔法、显微注射法等。

Leagene Lipofect 脂质体转染试剂(Lipofect Transfection Reagent)是一种新型的阳离子脂质体转染试剂，适用于把质粒、siRNA 或其它形式的核酸包括DNA 、RNA 、寡核苷酸以及核酸蛋白复合物或带负电荷的蛋白转染到真核细胞中。

Leagene Lipofect 脂质体转染试剂对于常见的哺乳动物细胞具有较高的转染效率，较好的重复性，且操作简以及单细胞毒性较小，可用于贴壁细胞和悬浮细胞。

Lipofect 脂质体转染试剂使用方法与Lipofectamine ® 2000 Reagent 极为相似。

该转染试剂转染细胞时， 基本不受细胞培养液中的血清和抗生素的影响，即可以在血清和抗生素存在的情况下进行细胞转染。

但为了取得最佳的转染效果，推荐转染时使用不含抗生素的含血清的细胞培养液。

组成：自备材料：1、 胰蛋白酶消化液2、 完全培养液和不完全培养液3、 PBS操作步骤(仅供参考)：(一)DNA 转染：1、 (以12孔板为例)在转染前18～24h 用胰蛋白酶消化培养细胞，取适量对数期细胞转移至12孔板中，并将细胞培养板置于CO 2 培养箱培养，待细胞密度达到即可进行转染。

后续操作步骤均按12孔板计算，如果转染器皿不同，请按比例自行调节用量。

2、 在加入待转染的DNA 之前2～4h ，加入不含抗生素的完全培养液，置于CO 2培养箱培养。

也可使用含有血清并含有抗生素的新鲜培养液，但抗生素使某些细胞转染后出现一定的细胞毒性。

3、 配制转染工作液：取两个无菌离心管，分别加入不含抗生素和血清的培养液，取其中一编号 名称CZ0002 CZ0002 CZ0002 StorageLipofect 脂质体转染试剂 0.5ml 1ml 5×1 ml 4℃使用说明书 1份离心管加入DNA，轻轻混匀；取另一离心管加入Lipofect脂质体转染试剂，轻轻混匀。

上海常用转染试剂配方
转染试剂是在细胞培养研究中必不可少的试剂。

它将核酸或蛋白质带入目标细胞中，以达到研究目的。

本文将介绍上海常用的几种转染试剂配方。

1. Lipofectamine 2000转染试剂
Lipofectamine 2000是一种常用的脂质体转染试剂，其用途广泛且转染效率高。

其配方如下：
无血清培养基：500μL
要转染的DNA：2μg（根据实验需要调整）
将Lipofectamine 2000试剂与无血清培养基混合，放置5分钟，再加入要转染的DNA 液体，混合后放置15分钟，滴加在细胞上。

2. PEI转染试剂
聚乙烯亚胺（PEI）是一种阳离子高分子，由于其能够形成与DNA负电荷的缔合物，使其成为一种有效的转染试剂。

PEI转染试剂可用于转染细胞以获得较高的转染效率。

其配方如下：
PEI试剂：4μg/μL，将其与等体积的无血清培养基混合。

3. PolyJet转染试剂
PolyJet是一种高效、无毒、具有低细胞损伤的DNA转染试剂。

其配方如下：
4. FuGENE HD转染试剂
细胞的转染操作应该注意以下几点：
1. 转染时间应该控制在半小时至1小时内，过长或过短都会对转染效率造成影响。

2. 转染的DNA浓度应该适当，过高的DNA浓度会对细胞产生毒性。

3. 转染试剂与DNA的混合比例也应该适当，过多或过少都会导致转染效率降低。

总之，合理选择转染试剂和配方，并严格按照操作步骤进行转染可以提高实验的可靠性和成功率。

Lipofectamine2000细胞转染protocol1、预处理：转染前一天，换液，胰酶消化细胞并计数，25c㎡的培养瓶中长满细胞可达5×10^6/mL，取3-4×10^5细胞铺在2mL的培养基中（不含双抗含血清），使其在转染时密度为90-95%（张老师做法：在细胞传代时用1mL胰酶消化，再加入4mL培养基（不含双抗含血清）中和。

800rpm，离心5min，5Ml培养基（不含双抗含血清）吹散后计数，留取1mL继续培养，其余按所需分装到4个6孔板，没孔加培养基至2mL）。

2、质粒DNA配制：每孔2ug质粒DNA，用无血清双抗OPTI-MEN培养基稀释至250ul；多孔可批量配制。

质粒DNA配好后，涡旋，瞬离。

3、Lipofect配制：每孔5ul lipofect试剂，用OPTI-MEN培养基稀释至250ul温和混匀，温室放置5min；多孔可批量配制。

4、将稀释的质粒DNA和Lipofect，混合在一起（配制后30min内混合），室温保温20min，保证每孔总体积500ul。

溶液可能变浑浊，但不影响转染。

（DNA-Lipofect复合物室温下6小时内保持稳定）。

5、将前天孵育的6孔板取出，处理细胞。

用移液管吸去上清（注意从边缘吸取，尽可能不要吸去贴附细胞）。

6、每孔2mLPBS清洗1遍，注意移液管要对着边缘加液，不可吹起细胞。

7、每孔用1mLDMEM清洗2遍。

8、每孔先加入OPTI-MEN0.9mL，再加500ulDNA-Lipofect复合物，摇动培养版，轻轻混匀（加质粒DNA时注意不要碰管壁）。

9、置入CO2培养箱，在6h后更换培养液，弃去上清，加入2mL不含双抗的DMEM。

10、再次置入CO2培养箱，72小时后，吸收上清至50mL离心管。

500转，10min 离心，取上清1mL/管（1.5mL进口EP管）分装标注，冻存-80°。

另取200ul 至2mLEP管检测HBsAg定量。

CAT. NO. 11668-027 Size:CAT. NO. 11668-019 Size: ml4℃储存（不要冻存）说明：Lipofectamin TM2000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 TM2000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%。

脂质体2000描述脂质体2000是专门用于将核酸转染进入真核细胞的一种形式，我们提供下列的建议：在多种细胞和培养板中都有很高的转染效率。

在网上列出的细胞类型中都获得了较高的转染效率。

核酸-脂质体2000的复合物可以直接加入到细胞培养液中，无论是有血清还是无血清的情况。

转染之后不用移除复合物或者更换培养液，但是复合物应在转染后4-6h移除。

转染的一些重要指导DNA转染使用page3我们建议使用Opti-MEM I reduced serum 培养基来稀释脂质体2000和核酸。

转染时不要向培养基中加入抗生素。

在实验过程中要保持一样的条件DNA转染使用下列方法在24孔板内转染DNA进入哺乳动物细胞。

对于其他的板子，可以参考page4.所有的数量和体积都是基于一个孔的剂量。

准备复合物使DNA（ug）：脂质体（ul）的比例为1:2到1:3。

转染时细胞密度较高可以获得高的效率，高的表达水平，以及使毒性降低。

条件的选择很重要。

1.贴壁细胞：在转染前一天，向24孔板内接种0.5-2*105个细胞，使得细胞在转染时融合率达到90-95%。

切记使用不含抗生素的培养基。

2.对于每一个转染样品，按照如下方法准备复合物：A．使用无血清的培养基将DNA稀释为50ul，温和混匀。

B．使用前混匀脂质体2000，然后用培养基将一定量的脂质体2000稀释为50ul。

室温孵育5min。

进行到c时间不超过25minC．5min的孵育后，混合稀释的DNA和稀释的脂质体2000（总体积为100ul）。

温和混匀，室温孵育20min（溶液呈现云雾状）。

混合物在室温可稳定存在6h。

3.向含有细胞和培养液的板子中每孔加入100ul的复合物，通过敲打板子和来回晃动使其混合均匀。

4.细胞孵育18-48h后可检测转染情况。

培养液可在转染4-6h后更换。

5.对稳定的细胞系：传代细胞在转染后以1:10的比例加入新鲜的培养液。

第二天加入选择培养基。

优化转染为了获得高的转染效率和低毒性，通过调整细胞浓度和DNA:脂质体2000的比例来优化转染条件。

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 protocolSKBR349 1.5 x 105 2Vero86 8 x 104 2Rat Hepatocytes50 1.25 x 105 1.5对于hela细胞，最好在转染前更换成无血清培养基Rat 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 MSDS24孔板的DNA用量REFERENCES(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 TM 2000之马矢奏春创作CAT. NO. 11668-019 Size: 1.5 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。

在上有转染步调，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：）（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，在实验过程中包管相同的接种条件。

4.为防止细胞死亡，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，因此有需要检测一下无血清培养基和Lipofectamine TM 2000的相容性。

转染步调（用于DNA）：依照如下步调在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尺度。

步调中均依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500μ×105个细胞，以包管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。