Lipofectamine 2000 Transfection Reagent
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原理
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。
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。
lipofectamine2000_man
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
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
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
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转染操作步骤
.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。
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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.。