如何准备转染所需的质粒DNA转染效率受到诸多因素的影响，除了细胞、培养基和载体等影响因素外，另外一项非常重要的因素便是DNA的质量。为了比较不同厂家的转染效率，我们分别从三家不同的供应商购买了质粒制备试剂盒来制备pEGFP-N3质粒DNA，并通过不同的方法将制备的pEGFP-N3质粒转运至NIH-3T3细胞。pEGFP-N3质粒分别通过Endofree Maxi Piasmid Kit(Qiagen),PerfectPrep Endofree Piasmid Maxi Kit(5 PRIME,inc)及NucleoBond DNA Maxi Kit(MACHEREY-NAGEL GmbH Co.KG)制备，我们测定了230,260,280及320nm的吸光值检测DNA的质量。通过上述三种方法制备的DNA质量如下：MACHEREY-NAGEL＞5Prime＞Qiagen。pEGFP-N3由PolyJetDNA转染试剂运至NIH-3T3细胞。转染效率的高低与DNA的质量相符，由MACHEREY-NAGEL纯化DNA对应的转染效率最高，而由Qiagen纯化DNA对应的转染效率最低。因此，MACHEREY-NAGEL GmbH Co.KG的质粒制备试剂盒被认为是制备转染级别DNA的最佳选择。How to prepare transfection grade plasmid DNATransfection efficiencies are affected by a variety of different parameters. Besides factor such as cell culture, medium and vectors, one of most critical parameters is DNA quality. We prepared pEGF-N3 plasmid DNA by large plasmid preparation kits from three different vendors and tested transfection efficiencies by delivering pEGFP-N3 DNAs prepared by different methods to NIH-3T3 cells.We prepared pEGFP-N3 plasmid with Endofree Maxi Plasmid Kit (Qiagen), PerfectPrep Endofree Plasmid Maxi Kit (5 PRIME, Inc.) andNucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH Co. KG). The DNA quality was determined by measuring absorbance at 230, 260, 280 and 320 nm by spectrometry.The purity of DNA prepared by the three methods is as follows:MACHEREY-NAGEL 5 Prime Qiagen.Then pEGFP DNA was delivered with PolyJet (Cat # SL100688) DNA transfection reagent to NIH-3T3 cells per manufacturer s protocol. In accordance with DNA purity results, we found that DNA fromMACHEREY-NAGEL gave the besttransfection efficiency while Qiagen DNA gave the worst efficiency. Therefore plasmid preparation kit fromMACHEREY-NAGEL GmbH Co. KG is confirmed to be the best choice to prepare tansfection grade DNA.表皮细胞的转染表皮细胞广泛遍布于身体，正常的表皮细胞较难转染，尤其是使用基于脂质体技术的转染试剂。我们使用电转(Amaxa)方法转染正常人的结肠表皮细胞并得到了65%的GFP标记细胞。非常感谢SignaGen,现在我们使用GenJet Ver II可以成功转染正常人的结肠表皮细胞并且转染效率显著提高至75%。如下是使用GenJet Ver II(Cat#SL100489)转染表皮细胞的简单步骤：1、转染时确保表皮细胞达到85%的融合度，并且保证细胞新鲜、健康。2、对于6孔板，用不含血清的DMEM分别稀释1.0 g,DNA及3.0 l,GenJet Ver.II（Cat＝SL100489）.将稀释好的转染试剂加入DNA中，室温下放置15分钟以形成转染复合物。3、将转染复合物直接加入表层细胞中：6孔板，每孔含1.0ml培养基，在血清/抗生素存在下，转染进行。4、在转染进行5小时后，清除转染复合物并换成正常生长培养基。5、转染后的24-48小时，检测转染基因的表达情况。Transfection of epithelial cells.Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix. Normal epithelial cell is extremely hard to transfect especially to liposome based transfection reagents. We ever used electroporation (Amaxa) to transfect normal human colonic epithelial cells and got 65% GFP+ cells. Thanks to SignaGen, now we have successfully transfectednormal human colonic epithelial cell with GenJet Ver. II with up to 75% GFP+ efficiency. The following is a brief protocol for transfecting epithelial cell with GenJet Ver. II (Cat # SL100489):1. Grow epithelial cell ~85% confluency at time of transfection. Epithelial cells must freshly prepared and healthy.2. For 6-well plate, dilute 1.0 g of DNA and 3.0 l of GenJet Ver. II (Cat # SL100489) per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes.3. Add transfection complex to epithelial cells directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.4. Remove transfection complex 5 hours after transfection and change back to normal growth medium.5. Check transgene expression 24~48 hours post transfection.针对特定细胞选择更有效的实验步骤GenJet Ver.II，LipoD293及PolyJet针对哺乳动物细胞的DNA体外转染试剂，均为您提供两种不同的转染步骤 一般步骤及高级步骤，这两种步骤分别针对不同的哺乳动物细胞。高级步骤主要针对难转的哺乳动物细胞，例如：MDCK，MDA-MB231，Caco-2等细胞。使用一般的转染步骤若是转染效率低于5%，那么使用高级转染步骤则可以将转染效率提升至60%。如何选择转染步骤则取决于您的细胞特性。对于诸如HEK293，Hela，CHO，3T3，COS，HepG2，LNCaP，PC3，PC12，U2-OS，L929，MCF-7及Huh-7等常用细胞，一般的实验步骤就能得到满意的转染结果。对于诸如MDCK，MDA-MB231，Caco-2，SaoS-2及HUVEC等难转细胞，可以选用高级转染步骤。针对您的细胞，若是您尚不明确选择哪种转染步骤，我们建议您可先尝试一般步骤，如果您使用一般步骤得到的转染效率低于5%，那么您的细胞比较难转，您可尝试高级转染步骤。Choose a more efficient protocol for your specific cell.GenJet Ver. II, LipoD293 and PolyJet DNA in vitro transfection reagent all offer two protocols for transfecting mammalian cells--------a general protocol and an advanced protocol. The two protocol are written for transfecting different types of mammalian cells. The advanced protocol which involves a shaving cell technology is for very hard to transfect mammalian cells like MDCK, MDA-MB231, Caco-2, etc. The general protocol usually gives less than 5% efficiency while advanced protocol can get up to 60% efficiency on these problematic cells.The principle to decide which one protocol is used to your cell is highly dependent upon the nature of your cell. For commonly used cells like HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929, MCF-7 and Huh-7, be sure to use general protocol which usually gives very good transgene expression while the advanced protocol does not help. For very hard to transfect cells like MDCK, MDA-MB231, Caco-2, SaoS-2 and HUVEC, follow the advanced protocol by trypsinizing these adherent cells first followed by incubation of the cell pellet with transfection complex. If you do not know for your specific cells how to choose the more efficient protocol, we suggest you try general protocol first. If you get less than 5% efficiency with the general protocol, then your cell is hard to transfect in nature and you can consider trying advanced one.如何优化PolyJet转染试剂我们使用PolyJet DNA转染试剂转染表皮细胞及Raw267.4细胞非常有效，并且毒性很小。对于如何更好的优化PolyJet转染试剂，我乐意分享以下几点：1、DNA的质与量。DNA的纯度对于转染实验至关重要。由E Coli制备的DNA，A260/280必须达到1.80甚至更高。对于6孔板，通常每孔~1.0 g DNA足矣。其他规格的细胞培养皿，可以根据表面积适当调整DNA含量。2、PolyJet/DNA的比率。对于表皮及Raw267.4细胞的转染，我们将PolyJet/DNA的比率固定在3:1，并且得到了满意的结果，没有为寻找更合适的比率问题而烦恼。3、稀释。DNA及PolyJet的稀释一定不要含有血清。我们使用的是不含血清的高糖DMEM培养基，效果很好。请不要选择Opti-MEM，因为它可以影响转染复合物的形成，因此，千万不要使用Opti-MEM来稀释质粒及PolyJet。4、转染中可以含有血清。我们在含有血清/抗生素及不含二者的情况下分别进行可转染实验，没有发现两种情况下转染效率有什么差别。因此，血清/抗生素对PolyJet转染试剂没有什么影响。Some technical tips for optimizing PolyJet reagent.PolyJet DNA Transfection Reagent is very efficient to transfect epithelial and Raw 267.4 cells in our hands without significant toxicity. Here I would like to share several technical points for better using PolyJet transfection reagent.1. DNA amount and quality. DNA purity is essential for high efficiency. DNA prepared from E Coli. must be 1.80 at A260/280 or higher. For 6-well plate, ~1.0 g of DNA per well is usually good enough. For other cell culture format, just adjust DNA amount per culture dish\'s surface area.2. Ratio of PolyJet/DNA. For epithelial and Raw 267.4 cells, we locked the ratio at 3:1 with excellent results and never bothered to find the optimal ratio.3. Diluent. Diluent used to dilute DNA and PolyJet reagent must be serum free. Serum-free DMEM medium with high glucose works for us. Opti-MEM is NOT a good choice because it may affect formation of transfection complex. So never use Opti-MEM as dilute.4. Transfection with serum. We performed transfection with or without serum/antibiotics and failed to find any difference in efficiency. Therefore, PolyJet reagent is NOT interfered by serum/antibiotics.如何优化LipoD293转染试剂LipoD293DNA转染试剂是脂质体DNA转运工具的升级版本。我们实验室使用LipoD293DNA转染试剂成功转染了HepG2，LNCaP，CHO及HEK293细胞。接下来，我们乐意就如何提高转染效率，降低毒性等方面的小技巧同大家分享。1、LipoD293/DNA的比率。尽管合适的比率由细胞类型决定，但是使用LipoD293/DNA，3:1的固定比率通常能得到很好的转染效率。2、每孔中DNA的含量。对于24孔板来说，我每孔使用的0.2到0.5 gDNA。太多的DNA（例如每孔1.0 g）没有必要，并且会产生较高的毒性。其他规格的细胞培养器皿，可以根据表面积适当调整DNA含量。3、DNA及LipoD293的稀释。一定不要使用含有血清的培养基来稀释二者。高糖的Plain DMEM培养基是不错的选择，但是，高糖并不是很重要。千万不要使用Opti-MEM（invitrogen生产）！Opti-MEM可以影响转染复合物的形成。我的同事没有认识到该点的重要性，错误地使用了Opti-MEM，导致转染效率低下。4、血清/抗生素的存在与否对转染没有影响。目前，我们正在使用的哺乳动物细胞通常是用LipoD293进行转染的，血清/抗清素对于转染效率没有影响。与其他依赖于脂质体的转染试剂不同，LipoD293不受血清/抗生素的影响，因此您不必担忧转染中含血清培养造成的高细胞死亡率问题。5、转染后更换培养基，对于我们正在研究的哺乳动物细胞，通常，我们是在转染后24小时更换培养基，没有必要在转染后5小时更换培养基。Some points which are important but ofetn neglected for optimizing LipoD293 reagent.LipoD293 DNA Transfection Reagent is an enhanced version of liposome DNA delivery tool. Our lab has been using LipoD293 DNA transfection reagent for transfecting HepG2, LNCaP, CHO and HEK293 cells with very good lucks.Now we like to share some technical points which I think are important for maximum efficiency and minimal toxicity and often ignored:1. Ratio of LipoD293 reagent/DNA. While the optimal ratio is cell type dependent, the ratio of reagent/DNA locked at 3:1 often gives excellent efficiency. Our labs has been transfecting HepG2, LNCaP, CHO and HEK293 with LipoD293 reagent at 3:1 ratio with very good efficiency.2. DNA amount per well. For 24-well plate, I used 0.2 to 0.5 g of DNA per well. Too much DNA (eg, 1.0 g per well) is unnecessary and might lead to high cytotoxicity. For other cell culture format, adjust DNA amount per culture dish\'s surface area.3. Diluent for diluting DNA and LipoD293 reagent. Diluent must be serum free. Plain DMEM medium with high glucose is usually very good. High glucose seems not that important. Never use Opti-MEM (from Invitrogen)! This is very important \'cause my colleagues often failed to recognize importance of the diluent and misused Opti-MEM, leading to suboptimal efficiency. Per our initial test, Opti-MEM is able to disrupt formation of transfection complex.4. Transfection with or without serum/antibiotics. For all mammalian cells we are currently working on, we always perform transfection with LipoD293 reagent in the presence of serum (10% FBS)/antibiotics without compromising any efficiency, thus greatly simplifying our transfection procedures. Unlike other liposome based reagent, LipoD293 reagent is usually NOT interfered by serum/antibiotics, so do not bother to perform transfection in serum-free medium which otherwise results in high cell death.5. Medium change post transfection. We usually change medium 24 hours after transfection. Medium change 5 hours post transfection is not necessary for all the mammalian cell we are currency using.转染L929细胞的简单步骤L929是小鼠成纤维细胞瘤细胞株，经常被用来检测TNF-alpha及TNF-beta。对TNF的处理经常会引发细胞凋亡及死亡，因此L929细胞经常被用作免疫分析。但是，转染L929细胞非常难，尤其使用基于脂质体技术的转染试剂，我们使用GenJet VerⅡ及PolyJet转染L929细胞获得了75%的转染效率，简单的实验步骤如下。1、转染时确保L929细胞达到80%的融合度，细胞必须健康并且传代不能超过9代。2、对于6孔板，用不含血清的DMEM分别稀释1.0 g，DNA及3.0 l，Genjet VerⅡ或PolyJet转染试剂。将稀释好的转染试剂加入DNA中，室温下放置15分钟以形成转染复合物，其它规格的细胞培养器皿，可以根据表面积适当调整DNA含量。3、将转染复合物直接加入L929细胞中；6孔板，每孔含1.0ml培养基，在血清/抗生素存在下,转染进行。4、转染后的24~48小时，监测转染基因的表达情况。Brief procedures for transfecting L929 cell.L929 is a murine aneuploid fibrosarcoma cell line which is often used to assay TNF-alpha and TNF-beta. Treatment with TNF initiates apoptosis and subsequent cell death, therefore L929 cell is often used for immunological assays. However L929 cell is hard to transfect, especially resistant to liposome based transfection method. We have been using GenJet Ver. II and PolyJet to transfect L929 cell and got up to 75% efficiency. The brief procedures are described below for transfecting L929 cells with GenJet and PolyJet.1. Grow L929 cell to ~80% confluency at the day of transfection. L929 cell must be healthy and less than 9 passages for maximum efficiency.2. For 6-well plate, dilute 1.0 g of DNA and 3.0 l of GenJet Ver. II or PolyJet reagents per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes. For other format of cell culture formats, scale down or up per the surface area of culture dish.3. Add transfection complex to L929 cell directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.4. Check transgene expression 24~48 hours post transfection.选择GenJetTM，LipoD293TM PolyJetTMDNA转染试剂稀释溶液的小技巧选择何种稀释液稀释DNA及转染试剂，对于制备有效的转染复合物至关重要。除了温度，孵育时间外，稀释液的性质对于制备高效的转染复合物亦非常重要，同样影响DNA转染效率。根据我们的实验数据，使用合适的稀释液得到的转染效率是使用错误稀释液转染效率的至少50倍。更为重要的是，实验者总是忽视稀释液的重要性，甚至一直未曾意识到正确的稀释液对形成有效转染复合物的重要性。如下实验技巧可以帮助您为GenJetTM，LipoD293TM PolyJetTMDNA转染试剂选择合适的稀释液。1、为了制备更有效的转染复合物，稀释液中千万不要含有血清、蛋白。2、尽量使用接近细胞培养基成分的稀释液，例如：若是你使用DMEM（supplemented with serum and antibiotics）培养HEK293细胞，那么，最合适的稀释液是不含血清、抗生素的DMEM：若是您使用RPM1-1640（supplemented with serum and antibiotics）培养Hela细胞，那么最合适的稀释液是不含血清、抗生素的RPM1-1640。3、请勿使用含有未知成分的培养基。例如，切勿使用Opti-MEMTM稀释GenJet 、PolyJet 及LipoD293 DNA转染试剂。根据我们的实验数据，若是使用Opti-MEM稀释转染试剂及DNA，那么用于HEK293，Hela，CHO及NIH 3T3细胞的转染效率至少减少50%。Opti-MEMTM可能含有较少的血清，导致转染效率的大幅降低。4、不含血清的高糖DMEM是很好的选择，可以与其他的生长培养基兼容，如果未能得到较高的转染效率，您可能会考虑更换稀释液，若是您不明确如何选择合适的稀释液，建议您初次可以尝试不含血清的高糖DMEM，您应该会得到满意的转染效率。Tricks in choosing diluent for GenJet , LipoD293 PolyJet DNA transfection reagents.For efficient formation of transfection complex, to choose an appropriate diluent which is used for diluting DNA transfection reagent and DNA is essential. In addition to temperature, incubation time, the nature of diluent is very important for complete formation of transfection complex, thus significantly affecting DNA transfection efficiency. Per our validation data, an appropriate diluent can be to 50 times more efficient than a wrong diluent. What\'s more important is researchers often neglect the importance of diluent and fail to recognize role of diluent in the process of transfection complex formation.Follow the following technical tips to choose the proper diluent for GenJet , PolyJet and LipoD293 DNA transfection reagents:1. For complete formation of transfection complex, the diluent must be serum-free and protein free.2. Try to use a diluent which is closest to the composition of cell culture medium. For instance, if you are using DMEM (supplemented with serum and antibiotics) to grow HEK293 cells, the best diluent is serum-free and antibiotics-free DMEM. If you are using RPMI-1640 Medium (supplemented with serum and antibiotics) to grow Hela cells, the best diluent is serum/antibiotic-free RPMI-1640 medium.3. Never use a medium which contains unknown components. For example, never use Opti-MEM to dilute GenJet , PolyJet and LipoD293 DNA transfection reagents. Per our validation data, diluting our transfection reagents and DNA with Opti-MEM at least sacrificed 50% transfection efficiency on HEK293, Hela, CHO and NIH 3T3 cells. Also Opti-MEM may contains low level of serum which will greatly compromise transfection efficiency.4. Basically serum-free DMEM with high glucose is pretty good choice. It is very easy accessible and compatible with other cell growth mediums. If you have trouble with lower transfection efficiency, you may think about changing diluent to serum-free DMEM with high glucose. If you do not know how to choose a proper diluent, you can try serum-free DMEM with high glucose first which usually gives you very good transfection efficiency.报告基因检测的精明之选 LipoD293DNA转染试剂使用lipoD293转染试剂来转染哺乳动物细胞（比如Hela 、PC3），以期检测荧光酶报告基因，同其他转染试剂相比较，您会得到更多的荧光酶读数，更少的细胞死亡率。使用unsupplemented RPM11640/DMEM替代Opti-MEM培养基来稀释lipoD293及DNA（luciferase reporters），您会得到更高的荧光酶读数（提高一倍），并且能节省更多的经费。如下是实验过程中的几个实验要点：1、使用相同类型的培养基（but unsupplemented）来稀释lipoD293试剂及luciferase reporterDNA（例如，若是细胞是用RPM11640-based培养基培养的，那么可选用unsupplemented RPM11640作为稀释溶液）；2、转染前及转染后，没有必要更换培养基；3、将稀释的lipoD293试剂加入稀释的DNA，混匀，在室温下孵育15-20分钟。Smart choice for reporter assay with LipoD293 DNA transfection reagent.Use lipoD293 DNA transfection reagent for transfection of multiple mammalian cell lines (such as Hela and PC3) with luciferase reporters, you will get much higher luciferase readings than other transfected reagents used on our lab for luciferase reading, but observe little cell death in transfected cells in our condition!Use unsupplemented RPMI1640 / DMEM instead of Opti-MEM medium to dilute both lipoD293 and DNA (luciferase reporters), you will get further higher luciferase readings (one-fold increase) and save more money !Several key points in the protocol:1) Use the same type of culture medium (but unsupplemented) for diluting lipoD293 reagent and luciferase reporter DNA( for example, if the cells are cultured in RPMI1640-based medium, then use unsupplemented RPMI1640 as dilution medium);3) No need to change medium either before or after transfection;4) Add diluted lipoD293 reagent to diluted DNA and then incubate the mixture at room temperature for 15~20 minutes;5) Make lysates for luciferase assay 16~48hrs post transfectionsiRNA体外转染GenMuteTM siRNA体外转染的优化GenMuteTM siRNA转染试剂（Cat#SL100568）是市场上最有力的siRNA传递工具之一。最佳的siRNA浓度范围是1.0nM到10nM，过多的siRNA可能导致沉默效果差的 洪水效应 。我们实验室已经使用GenMuteTM转染试剂成功敲除了内源表达的生长因子。以24孔板为例，如下步骤将对如何优化GenMuteTM转染试剂做一指导：至于其他规格器皿的培养的细胞，烦请参考GenMuteTM的实验说明。1、在转染前的30~60分钟，更换培养基，并在每孔中加入0.5ml的完全培养基（含血清和抗生素）。2、将2.0pmol siRNA（5.0 M x 4.0 l）加入到盛有200 l GenMute转染缓冲液（Cat#SL100572）的无菌管中进行稀释，室温下静置5分钟。3、加入2.0 l GenMute转染试剂，混匀，在室温下放置15分钟形成转染复合物。请注意：室温下，转染复合物的静置时间勿超过25分钟。4、取50 l、25 l、12.5 l转染复合物分别加入细胞中（复孔），siRNA的终浓度各自达到10nM、5.0nM、1.25nM。5、转染24~48小时后，检测4个不同siRNA浓度下的转染效果，选择出最佳的转染条件。Optimization of GenMute reagent for siRNA silencing.GenMute siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations range from 1.0 nM to 10 nM. Excessive siRNA may lead to flooding effect with sub-optimal silencing. Our lab has been using GenMute reagent to knock down endogenously expressed growth factors with very good luck. The following procedures will guide to optimize GenMute reagent for best silencing in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute reagent.1). Change medium and add 0.5 ml of complete medium each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.2). Dilute 20 pmol siRNA (5.0 M x 4.0 l) into 200 l of GenMute transfection buffer (Cat # SL100572) in a sterile tube and let\'s sit at RT for 5 minutes.3). Add 2.0 l GenMute reagent, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.4). Add 50 l transfection complex to your cells directly in duplicate (final 10 nM siRNA), 25 l complex to another 2 wells (final 5.0 nM siRNA), 12.5 l to 3rd 2 wells (final 2.5 nM siRNA) and 6.25 l to 4th 2 wells (final 1.25 nM siRNA).5). Check silencing effect in the 4 different siRNA concentrations 24~48 hours post transfection and choose the best transfection conditions.如何优化GenMuteTM转染试剂，提高siRNA/DNA共转染效率GenMuteTM siRNA转染试剂（Cat#SL100568）是市场上最有力的siRNA传递工具之一。siRNA/DNA共转染时，siRNA的最佳浓度范围是0.5 M到10 M，过多的siRNA可能导致沉默效果差的 洪水效应 ，切勿使用超过20 M的siRNA。以24孔板为例，如下步骤将对如何优化GenMuteTM转染试剂做一指导；至于其他规格器皿的培养的细胞，烦请参考GenMuteTM的实验说明。1、在转染前的30-60分钟，更换培养基，并在每孔中加入0.5ml的完全培养基（含血清和抗生素）。2、将0.5 g DNA各自加入到分别盛有50 l GenMute转染缓冲液（Cat#SL100572）的4个无菌管中进行稀释，接下来分别对4管中的siRNA进行系列稀释 将5.0pmol siRNA加入到第一管中，2.5 pmol siRNA加入到第二管中，1.25 pmol siRNA加入到第三管中，1.25 pmol siRNA加入到第四管中。室温下静置5分钟。3、取3.0 l GenMuteTM转染试剂，分别加入到稀释好的siRNA/DNA管中，混匀，室温下静置15分钟以形成转染复合物。请注意：室温下，转染复合物的静置时间勿超过25分钟。4、将如上制备的转染复合物分别加入24孔板中连续的4个孔中。5、转染24-48小时后，分别检测4孔中的沉默效果并选择出最佳的转染条件。Optimization of GenMute reagent for siRNA/DNA co-transfection.GenMute siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations for siRNA/DNA co-transfection range from 0.5 nM to 10 nM. Excessive siRNA may lead to flooding effect which may comprise silencing effect, so never use siRNA higher than 20 nM. The following procedures will guide to optimize GenMute reagent for best siRNA/DNA co-transfection in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute reagent.1. Change medium and add 0.5 ml of complete medium to each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.2. Dilute 0.5 g DNA to each sterile tube of total 4 tubes with 50 l of GenMute transfection buffer (Cat # SL100572) followed by addition series diluted siRNA to each well of the total 4 tubes-------add 5.0 pmol siRNA to 1st tube, 2.5 pmols to 2nd tube, 1.25 pmols to 3rd tube and 1.25 pmols to 4th tube. Let\'s sit at RT for 5 minutes.3. Add 3.0 l GenMute reagent to diluted siRNA/DNA of each tube, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.4. For 4 consecutive wells of 24-well plate, add the transfection complexes which are prepared with same concentration of DNA (0.5 g per well) and different 4 concentrations of siRNA ranging from final 10 nM to 1.25 nM.5. Check silencing effect in the 4 wells 24~48 hours post transfection and choose the best transfection conditions.如何有效地降低PepMuteTM，GenMuteTM and PepMuteTM Plus siRNA转染试剂的毒性PepMuteTM，GenMuteTM 及 PepMuteTM Plus转染试剂，仅被发现在DMEM基础细胞培养基中偶有毒性，使用其他的培养基（含10%FBS及抗生素）培养细胞，将基本减少转染中的毒性。譬如，如果您发现有大量细胞死亡，您可能使用含10%FBS及抗生素的DMEM基础培养基。要消除毒性，您可以选择DMEM/F12或者RPMI 1640来替代DMEM作为基础培养基。至于为什么，到目前为止，我们也是不得而知，但是，它却有效地减少了毒性。试试吧！How to eliminate toxicity of PepMute , GenMute and PepMute Plus siRNA ReagentsPepMute , GenMute andPepMute Plus siRNA transfection reagents are found to be sometimes toxic ONLY with DMEM base cell culture medium. Using other cell culture mediums (supplemented with 10% FBS and antibiotics) to grow your cells will basically eliminate the transfection toxicity. For instance, if you find toxicity and high cell death, you are probablyusing DMEM base medium supplemented with 10% FBS and antibiotics to grow your cells. To remove the transfection toxicity, you can change DMEM to DMEM/F12 or RPMI 1640 as base medium.So far we have not figured out why, but it does minimize the toxicity. Try it out!