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fugene 4k 转染试剂中文说明书

2022版 CTM694原英文技术手册TM694中文说明书适用产品目录号：E5911和E5912FuGENE ®4K TransfectionReagent普洛麦格(北京)生物技术有限公司Promega (Beijing) Biotech Co., Ltd 地址：北京市东城区北三环东路36号环球贸易中心B座907-909电话：************网址：技术支持电话：400 810 8133技术支持邮箱：*************************CTM 6942022制作1所有技术文献的英文原版均可在/ protocols 获得。

请访问该网址以确定您使用的说明书是否为最新版本。

如果您在使用该试剂盒时有任何问题，请与Promega 北京技术服务部联系。

电子邮箱：*************************1. 描述 (2)2. 产品组分和储存条件 (2)3. 一般注意事项 (2)3. A. 转染试剂与DNA的比例 (3)3. B. DNA (3)3. C. 时间 (3)3. D. 血清 (3)3. E. 细胞培养条件 (3)3. F. 稳定转染 (3)4. 推荐操作步骤 (4)4. A. 细胞铺板 (5)4. B. FuGENE® 4K Transfection Reagent准备 (5)4. C. 一般转染操作步骤 (6)4. D. 稳定转染操作步骤 (8)4. E. 转染优化 (9)4. F. 报告基因活性和细胞健康的多重检测方案 (11)5. 疑难解答 (12)FuGENE® 4K Transfection Reagent普洛麦格(北京)生物技术有限公司Promega (Beijing) Biotech Co., Ltd 地址：北京市东城区北三环东路36号环球贸易中心B座907-909电话：************网址：技术支持电话：400 810 8133技术支持邮箱：*************************CTM 6942022制作21. 描述FuGENE® 4K Transfection Reagent是一个多组分，非脂质体试剂，用于将DNA高效、低毒地转染至多种哺乳动物细胞系中，无需在加入试剂-DNA复合物后更换培养基。

细胞转染步骤

细胞转染及其筛选1.传代：细胞于10cm盘消化后接种于6cm盘向6cm盘中加入3ml培养基,“米”字形摇晃。

当细胞生长到50％-70%时，根据细胞的生长速度可进行转染2.配置转染体系:(6cm盘)2。

4ug基因载体质粒；9ul转染试剂；200ul无血清培养基.混匀后室温静置10—15min孵育。

3.将6cm盘中的旧培养基弃去，换新的培养基3ml。

4.将配置好的转染体系加入到6cm盘中，混匀6-18h内换液。

5.12小时，换液后进行显微镜拍照观察。

6.再过6h加入G418，进行筛选(始终在6cm盘中进行）,使用G418浓度：600µg/mlG418的配置：取1gG418溶于1mlHEPES液中，加蒸馏水至10ml，过滤消毒4℃保存。

7.3-5天换液一次（换液不用PBS冲洗），此时仍要加G418，浓度不变,只到细胞呈单个细胞那种，当细胞死的过多时,可考虑药物浓度减半.筛选出稳定表达的cell。

8.挑克隆：1制备细胞悬液，细胞计数，用培养基稀释细胞到1个/10ul,在96孔板中加入培养基150ul/孔,在加入细胞悬液10ul/孔,待其逐渐增多后转入24孔板、6孔板、6cm 盘增殖。

2.伴随着细胞的生长,可能最后会变成细胞簇,故可以用黄枪头把细胞菌落挑出，放入24孔板里面。

9.单克隆鉴定:提取细胞RNA后，进行RT-PCR检测其表达量。

10.先做RT-PCR筛选一部分,在做western继续筛选一部分.所需物品:1。

转染试剂（Attractene Transfection Regent）；2.96孔板；3。

24孔板；4.6孔板；5.6cm盘；6。

G418 7. 1mlHEPES液。

各种转染试剂的中文转染方法

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

将细胞以1-3×105/2 ml接种于6孔板后孵育过夜将达到如此密度。

将FuGENE6 Reagent在室温孵育10-15分钟。

使用之前将FuGENE6颠倒混匀一下。

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

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

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

4. 室温孵育20分钟。

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

6. 将转染复合物加入细胞，混匀使之均匀分布。

7. 3-8小时后，加入血清或换成含血清的营养液。

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

细胞铺板在2 ml含血清，不含抗生素的正常生长的培养基中。

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

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

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

NOTE：若使用DMEM培养基，则需在5分钟内同稀释的DNA混合。

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

室温放置20分钟。

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

各种转染试剂中文说明

FuGENE6（Roche）转染步骤:转染前一天将细胞分至培养板，转染当天细胞应50-80％融合。

将细胞以1-3×105/2ml接种于6孔板后孵育过夜将达到如此密度。

将FuGENE6 Reagent在室温孵育10-15分钟。

使用之前将FuGENE6颠倒混匀一下。

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

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

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

4.室温孵育20分钟。

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

6.将转染复合物加入细胞，混匀使之均匀分布。

7.3-8小时后，加入血清或换成含血清的营养液。

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

细胞铺板在2ml含血清，不含抗生素的正常生长的培养基中。

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

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

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

NOTE：若使用DMEM培养基，则需在5分钟内同稀释的DNA混合。

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

室温放置20分钟。

5.（optional）将6孔板中的旧营养液吸出，用无血清培养基清洗两次。

加入2ml无血清配养基。

FuGENE HD转染试剂说明书

FuGENE ®HD Transfection Reagent1.What this Product DoesNumber of Transfection ExperimentsIn a typical experiment using HeLa or COS-1 cells, 1 ml of FuGENE ®HD Transfection Reagent can be used to perform up to three hundred transfections in 35-mm tissue-culture dishes, using 3 ␮l of reagent combined with 1 – 2 ␮g DNA per well. This is equivalent to over 6,000wells in a 96-well plate or 1,000 wells in a 24-well plate.L Optimal expression depends upon experimental conditions includ-ing cell type, passage history, confluence, seeding protocol, com-plex incubation time, serum batch, etc. The above amounts of reagents work well with HeLa or COS-1 cells. In other test systems,two- to three-fold higher amounts of reagent yield optimal levels of expression.FormulationFuGENE ® HD Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterile-filtered, and pack-aged in glass vials. It does not contain any ingredients of human or animal origin.Storage and Stability•FuGENE ® HD Transfection Reagent is shipped at +15 to +25°C.•Store FuGENE ® HD Transfection Reagent at +2 to +8°C, with the lid very tightly closed. The reagent is stable through the expiration date printed on the label when stored under these conditions.L FuGENE ® HD Transfection Reagent remains fully functional evenafter repeatedly opening the vial (at least five times over a two-month period) as long as the vial is tightly recapped and stored at +2 to +8°C between uses.L Do not store FuGENE ® HD Transfection Reagent below 0°C.Components may precipitate and alter results. If you accidently place the reagent at Ϫ20°C, briefly warm it to 37°C to dissolve any precipitate. It should function normally; however, do not return it to the freezer.Special HandlingN Always bring to room temperature and mix FuGENE ® HD Transfec-tion Reagent prior to use (vortex for one second or use inversion).N Do not aliquot FuGENE ® HD Transfection Reagent from the originalglass vials. Chemical residues in plastic vials can significantly decrease the biological activity of the reagent. Minimize the contact of undiluted FuGENE ® HD Transfection Reagent with plastic sur-faces.N Always dilute the reagent by pipetting directlyinto serum-free medium. Do not allow the FuGENE ® HD Transfection Reagent to contact the plastic walls of the tube containing the serum-free medium during the dilution step.N Do not use siliconized pipette tips or tubes.Additional Equipment and Reagents RequiredAdditional reagents and equipment required to perform transfection assays using FuGENE ® HD Transfection Reagent, but not provided,include:General Laboratory Equipment•standard cell culture equipment (e.g., biohazard hoods, incubators,microscope)•standard pipetters and micropipetters •vortex mixerFor Plasmid Preparation•purified plasmid stock (0.1 ␮g/␮l – 2.0 ␮g/␮l) in sterile TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer or sterile water•Genopure Plasmid Midi Kit*, Genopure Plasmid Maxi Kit*, or High Pure Plasmid Isolation Kit* can be used to prepare plasmid.For Verification of Vector Function •assay appropriate for transfected gene•G-418* or Hygromycin B* (optional; for stable transfection experi-ments)For Transfection-Complex Formation•Opti-MEM I Reduced Serum Medium, water, or serum-free medium •24-well plate to serve as test tube rack for FuGENE ® HD Transfec-tion Reagent vial•sterile polystyrene tubes or round-bottom 96-well platesCells Growing in Log Phase•select subconfluent cultures in log phase for preparation of the cell cultures for transfection•method to quantify cell number to reproducibly plate the same number of cellsApplicationFuGENE ® HD Transfection Reagent is a multi-component reagent that forms a complex with DNA, then transports the complex into animal or insect cells. Benefits of FuGENE ® HD Transfection Reagent include:•High transfection efficiency in many common cell types, including HeLa, NIH/3T3, COS-1, COS-7, CHO-K1, Hep G2, HEK-293, MCF7,and some insect cell lines. In addition, you will achieve excellent transfection efficiency in some cell lines (e.g., RAW) that are not transfected well by other reagents. Detailed transfection protocols and sample results are available at .•Demonstrates minimal cytotoxicity or changes in morphology when adequate numbers of cells are transfected, and eliminates the requirement to change media after the addition of transfection complex.•Suitable for transient and stable transfection.•Functions exceptionally well in the presence or absence of serum;eliminates the need to change media.To ensure the quality of cells to be transfected, Roche recommends using freshly-obtained, low-passage cell sines form ATCC ®. For more information please visit and bookmark .For the transient and stable transfection of animal and insect cellsCat. No. 04 709 691 0010.4 ml (up to 120 transfections)Version November 2007Cat. No. 04 709 705 001 1 ml (up to 300 transfections)Store at +2 to +8°CCat. No. 04 709 713 001 1)Mega-pack 5 × 1 ml (up to 1,500 transfections)Cat. No. 05 061 369 00110 ml (up to 3,000 transfections)Cat. No. 04 883 560 001Trial-pack1)The five vials are packaged together in one box with one pack insertFor life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.2.How to Use this Product2.1Before you BeginRequired Amount of FuGENE® HD Transfection ReagentFor initial optimization experiments, transfect a monolayer of cells that is 80 – 90% confluent in a six-well culture dish, using 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 ratios of FuGENE® HD Transfection Reagent (␮l) to DNA (␮g), respectively. For most cell types, these FuGENE® HD Transfection Reagent:DNA ratios provide excellent transfection levels.L Subsequent optimization may further increase efficiency in your particular application. In addition to varying the volume of FuGENE® HD Transfection Reagent, other parameters may be evaluated (see section 2.6, Parameters for Optimization, and sec-tion 3, Troubleshooting).Plasmid DNA•It is critical to accurately determine the plasmid DNA concentration using 260-nm absorption (estimates of DNA content based on the intensity of gel bands are not sufficiently accurate). Determine the DNA purity using a 260 nm/280 nm ratio; the optimal ratio is 1.8.•Prepare the plasmid DNA solution in sterile TE (Tris/EDTA) buffer or sterile water at a concentration between 0.1 ␮g/␮l and 2.0 ␮g/␮l. Cell Culture ConditionsMinimize both intra- and inter-experimental variance in transfection efficiency by using cells that are regularly passaged, proliferating well (best when in a log-growth phase), and plated at a consistent den-sity. FuGENE® HD Transfection Reagent is different from FuGENE® 6 Transfection Reagent regarding the optimal density of cells required for maximal expression with minimal negative effect; FuGENE®6 Transfection Reagent is formulated to work at low cell densities, whereas FuGENE® HD Transfection Reagent is formulated to work at higher cell densities. Cells must be healthy and free of mycoplasma and other contaminants.L If you have used FuGENE® 6 Transfection Reagent in the past, we suggest that you increase the plating density for initial tests using FuGENE® HD Transfection Reagent. For most cell lines, use the reagent at cell-plating densities at least twice that used with FuGENE® 6 Transfection Reagent to yield maximum protein expression. For most cell lines, cultures should be 80 – 90% con-fluent at the time of transfection. For contact-inhibited cell lines such as NIH/3T3, optimal results are obtained when cells are plated at lower densities.Other Media AdditivesIn some cell types, antimicrobial agents (e.g., antibiotics and fungicides) that are commonly included in cell-culture media may adversely affect the transfection efficiency of FuGENE® HD Transfection Reagent. If possible, exclude additives for initial experi-ments. Once high-efficiency conditions have been established, these components can be added back while monitoring your transfection results. Cell growth and/or transfection efficiency may be affected by variations in sera quality or media formulations.Verification of Vector FunctionOptimize transfection conditions with a known positive-control reporter gene construct prior to transfecting cells with a new vector construct:•Determine transfection efficiency with a reporter gene assay (CAT*,␤-Gal*, Luciferase*, SEAP*, or hGH*).•Sequence across the flanking vector insert regions to verify the integrity of your new construct.2.2Preparation of Cells for Transfection2.3Overview of Initial Transfection ExperimentAdherent and Suspension Cells in a Six-well Plate or 35-mm Culture DishFor initial optimization, test FuGENE® HD Transfection Reagent:DNA ratios of 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 (␮l for FuGENE® HD Transfection Reagent, and ␮g for DNA, respectively). The preparation of the com-plex for a single well of a six-well plate, or a 35-mm culture dish, is described in section 2.4. These ratios will function very well for com-monly used adherent cells and suspension cells. For your particular cell line and culture conditions you may find that ratios of 9:2, 10:2, 11:2, or 12:2 result in even greater expression. Try these ratios if you find the highest expression levels in the 8:2 ratio well.N Prepare the transfection complex in diluent that does not contain serum (e.g., Opti-MEM I Reduced Serum Medium), even if the cells are transfected in the presence of serum. For some cell lines, the complex may be formed in DMEM or sterile water.L For additional optimization tips, see section 2.6 and visit /fugene/hdRatio OverviewPreparation of a transfection complex that is sufficient for a 35-mm culture dish, or one well of a six-well plate, at six different ratios: Tab. 1: Preparation of transfection complex for a 35-mm culture dish.Cell Type ProcedureAdherentcellsOne day before the transfection experiment, trypsinizethe monolayer, adjust cell concentration, and plate thecells in the chosen cell-culture vessel. For most celltypes, plating 3 – 6 × 105 cells in 2 ml of medium in a35-mm culture dish (or six-well plate) overnight willachieve the desired density of Ͼ80% confluency atthe time of transfection. For cell lines with specialcharacteristics, such as contact-inhibited NIH/3T3cells, a lower plating density should be used. If usingculture plates of a different size, adjust the total num-ber of cells, starting volume of FuGENE® HD Transfec-tion Reagent, and the starting mass of DNA inproportion to the relative surface area (Table 2). SuspensioncellsUse freshly passaged cells at a concentration of 5×105/ml to 1 × 106/ml in 2 ml of medium in a 35-mmculture dish (or six-well plate). If using culture platesof a different size, adjust the total number of cells,starting volume of FuGENE® HD TransfectionReagent, and the starting mass of DNA in proportionto the relative volume (Table 2).Tubelabel(ratio)Diluent(␮l)FuGENE® HDTransfectionReagent (␮l)DNA(␮g)Comments3:210032Add the entire volume toa 35-mm culture dish oreach well of a six-wellplate, or 2 – 15 ␮l to eachwell of a 96-well plate.Suggested volumes fordifferent culture vesselsare included in Table 2. 4:2100425:2100526:2100627:2100728:2100822.4Transfection ProcedureNotes:L As with any experiment, include appropriate controls. Preparewells with cells that remain untransfected, cells with transfection reagent alone, and cells with DNA alone.L For stable transfection experiments, the complex-containingmedium should be left unchanged until the cells need to be pas-saged. At that time, include the appropriate selection antibiotics (G 418* or Hygromycin B*).L To prepare transfection complexes for different-sized containers orparallel experiments, proportionally change the quantity of all components according to the total surface area of the cell culture vessel being used (Table 2).L For ease-of-use when transfecting small volumes, as in 96-wellplates containing 0.1 ml culture medium per well, prepare 100 ␮l of transfection complex and add 2 – 15 ␮l to each well depending upon the cell type.L The optimal ratio of transfection reagent:DNA and the optimaltotal amount of complex may vary with cell line, cell density, day of assay, and gene expressed.L After performing the optimization experiment where several ratioswere tested, select a ratio in the middle of the plateau for future experiments.2.5Cotransfection ExperimentsSuggestionsFor cotransfection experiments with FuGENE ® HD Transfection Reagent, maintain the same total reagent:total DNA ratio as that used for a single plasmid in your system. Thus, the total amount of the plas-mid DNA should be equal to the amount of plasmid used in a single plasmid transfection.ᕡAllow FuGENE ® HD Transfection Reagent, DNA, anddiluent to adjust to +15 to +25°C. Vortex for one second or invert the FuGENE ® HD Transfection Reagent vial to mix.ᕢDilute DNA with appropriate diluent, for example, Opti-MEM IReduced Serum Medium, serum-free medium (without anti-biotics or fungicides), or sterile water to a concentration of 2␮g plasmid DNA/100 ␮l Opti-MEM (0.02␮g/␮l).L For insect cells, use sterile water as diluent. For other celllines, try sterile water or serum-free medium as an alterna-tive diluent.ᕣPlace 100 ␮l diluent, containing 2 ␮g DNA into each of six ster-ile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2.Recommendation : Use sterile polystyrene tubes or round-bottom, 96-well plates to form the transfection complex.L Due to manufacturer variability with release agents for96well plates, we suggest using tissue culture treated 96well plates to reduce variablity.ᕤForm the transfection complex by adding FuGENE ® HDTransfection Reagent to tubes containing diluted DNA :Pipet the FuGENE ® HD Transfection Reagent (3, 4, 5, 6, 7, or 8␮l) directly into the medium containing the diluted DNA with-out allowing contact with the walls of the plastic tubes.N To avoid adversely affecting transfection efficiency, do notallow undiluted FuGENE ® HD Transfection Reagent to come into contact with plastic surfaces (such as the walls of the tube that contains the serum-free medium) other than pipette tips. Do not use siliconized pipette tips or tubes.ᕥMix and incubate the transfection complex :Vigorously tap the tube or vortex for one to two seconds to mix the contents. If using a 96-well plate, place the plate on a rotat-ing shaker for 5 – 10 seconds. Incubate the transfection reagent:DNA complex for 15 minutes at room temperature.For some ratios and cell types, incubation is not necessary for optimal complex formation, while a longer incubation time is better for other cell types. Determine this for your particular cell line and the ratio you use.ᕦAdd the transfection complex to cell s:Remove culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection complex to the cells in a drop-wise manner or add below the surface of the medium. Swirl the wells or flasks to ensure distribution over the entire plate surface. Use of a rotating platform shaker for 30 seconds at low speed provides adequate mixing for 96-well plates.Once the FuGENE ® HD Transfection Reagent:DNA complex has been added to the cells, there is no need to remove and replace with fresh medium (as is necessary with some other transfection reagents).L In our experience, the exposure of most common laboratorycell types (COS-1, CHO-K1, HEK-293, HeLa, Hep G2, MCF-7) to the transfection complex until performance of the gene expression assay (24–48 hours later) does not affect the results. If you desire to transfect cells that are in serum-free medium during the transfection process, then replace the medium with serum-containing medium 3 – 8 hours after transfection, unless the cells normally grow in serum-free medium. ᕧIncubate cells and assay the results :Following transfection, incubate the cells for 18 – 72 hours prior to measuring protein expression. The length of incubation depends upon the transfected vector construct, the cell type being transfected, the cell medium, cell density, and the type of protein being expressed. After this incubation period, measure protein expression using an assay that is appropriate for your system.L If you observe low transfection levels or more than10 – 30% cell death, refer to section 3, Troubleshooting and /fugene/hd2.6Parameters for Optimization2.7Transfection of Adherent Cells Adapted for Suspension Growth•In some cases, adherent cells may be adapted for suspension growth, thus enabling the production of transiently transfected cells on a very large scale.•HEK-293 cells grown in suspension in serum-free medium that did not contain heparin or dextran sulfate produced significant amounts of protein following transfection.2.8Guidelines for Preparing FuGENE® HD Transfection Reagent:DNA Complex for Various Culture Vessel SizesThe starting volume and mass to add to the different culture vessels is based upon preparing a 100-␮l transfection complex as described in sec-tions 2.3 and 2.4. For best results, prepare a 100-␮l complex at different ratios and add varying amounts of each ratio when optimizing. The amounts below are based on the 100-␮l complex as prepared in sections 2.3 and 2.4.Suggested seeding density for adherent cells = 30,000 – 70,000 cells per cm2Suggested seeding density for suspension cells = 250,000 – 500,000 cells per mlTab. 2: Refer to the table below when setting up your transfection reac-tions. T hese are suggested seeding densities and are media, passage level, laboratory, and cell-line dependent. It is critical that log phase cultures are selected for subculture for the transfection experiments, and that cultures are seeded at the proper density for the transfection experiment. Observe cultures and plate them so that the monolayer is 80–90% confluent at the time of trans-fection. This must be determined empirically. For some cell lines, 60–80% con-fluency is sufficient. However, a contact-inhibited cell line, such as NIH/3T3, should be plated at lower confluence due to its growth characteristics.1) Scale up total volume for larger vessels.3.TroubleshootingParameter to beoptimizedProcedureFuGENE® HD Transfection Reagent:DNA ratio Form the transfection complex at several ratios: 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 10:2, and 12:2 (␮l FuGENE® HD Transfec-tion Reagent: ␮g DNA).In some systems, altering the ratio of FuGENE® HD Transfection Reagent to DNA can increase the level of protein expression.L It has been reported that for some plasmid preparations, a ratio of 2:2 yielded optimal results. This is unusual and may reflect some property of the plasmid preparation rather than a characteristic of the FuGENE® HD Transfec-tion Reagent.Amount of transfectioncomplex addedTry adding 200%, 150%, 75%, 50%, and 25% of the amount of 100-␮l transfection complex suggested in Table 2.Number of cells plated Plating more cells will overcome negative growth effects of excess transfection complex. For cells with special growth characteristics, such as NIH/3T3 cells, do not use this as the first parameter for optimization.Incubation time for the transfection complex to form Vary the length of incubation time for transfection-complex formation: add the complex to the cells immediately after the components are combined and mixed, and then at several intervals up to 40 minutes (i.e., 0, 15, 25, and 40 min-utes). We have observed that in some cell lines, the transfection-complex incubation time tends to have no effect on results when using higher ratios; however, results using lower-ratio-complexes varied depending on the incubation time for complex formation.Special tips for sensitive cell lines •Reduce the time of exposure to the transfection complex (2–3 hours maximum), then replace the medium.•Use the lower ratios, and allow the complex to form for a longer period of time (determine empirically for your cell line), then add lower amounts of the complex (50% or less of what was originally tested).Culture vessel Surfacearea(cm2)TotalvolumeofmediumSuggested seeding densitySuggested amount ofthe 100-␮l trans-fection complex toadd to each well (␮l)Final amount of FuGENE® HDTransfection Reagent (␮l) ineach well following addition ofsuggested amount of 100-␮ltransfection complexCells/wellAdherent cellsCells/wellSmall or suspensioncellsUsing the3:2 ratioUsing the8:2 ratio totalvolumevolume forlargerlow high low high96-well plate(1 well)0.30.110,00020,00025,00050,00050.150.424-well plate(1 well)1.90.550,000125,000250,000500,000250.752.012-well plate(1 well)3.8 1.0100,000250,000375,000750,00050 1.54.0 35-mm dish82200,000500,000500,0001,000,000100 3.08.06-well plate(1 well)9.42200,000600,000500,0001,000,000100 3.08.0 60-mm dish215500,0001,400,0001,250,0002,500,000250 1)7.520.0 10-cm dish55101,500,0003,500,0002,500,0005,000,000500 1)15.040.0 T-25 flask256700,0001,700,0001,500,0003,000,000300 1)9.024.0 T-75 flask75202,000,0005,000,0005,000,00010,000,000900 1)27.072.0Low trans-fection efficiency Poor quality orinsufficient quantityof nucleic acidsVerify the amount, purity, and sequence of nucleic acid.Perform a control transfection experiment with a commercially available transfection-grade plasmidpreparation.Chemical contaminants may be in the plasmid preparation. Avoid phosphate buffers until you havetested them in your system.L Endotoxins are reported to be cytotoxic to some very sensitive cell lines.Insufficient numberof cellsUse adherent cells that are at least 80% confluent. Low cell density results in fewer cells available totake up transfection complex, and excess complex may be cytotoxic; in addition, fewer cells yieldless protein.Too many cells orcells post log phaseWhen confluent cultures are subcultured, or cells are plated at too high a density, the cells fail to dividein the culture being transfected. This results in suboptimal expression.Suboptimal FuGENE®HD TransfectionReagent:DNA ratio,complex incubationtime, total amount oftransfection complexadded, or cell densityOptimize the FuGENE® HD Transfection Reagent:DNA ratio, complex incubation time, amount ofcomplex added to cells, and cell density, according to the following procedure:Day before transfection:Prepare two 96-well plates of cells at high and low seeding densities (see Table 2 for suggestions).Day of transfection:•Form 200 ␮l of transfection complex at ratios of 2:2, 3:2, 4:2, 5:2, 6:2. 7:2, and 8:2 (␮l transfectionreagent:␮g DNA) following the protocol in this pack insert (sections 2.3, 2.4) and doubling theamounts of all components.•As soon as the complexes are combined and mixed, add 10, 5, or 2.5 ␮l of each complex to one of3columns of cells in each 96-well plate (i.e., columns 2, 3, and 4). Leave all outer wells empty ascontrols.•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,5, or 2.5 ␮l of each complex to the next 3 columns (5, 6, and 7) of cells in each 96-well plate.•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,5, or 2.5 ␮l of each complex to the next 3 columns (8, 9, and 10) of cells in each 96-well plate.•Assay the plates 1–2 days later. Select the ratio, amount of complex, and time of transfection-complexincubation that resulted in optimal expression.•If optimal transfection occurs at the higher ratios, repeat this process using ratios of 6:2, 7:2, 8:2, 10:2,12:2, and 14:2. Add 5, 10, and 15 ␮l of complex. We have never successfully transfected cells using theratio of 2:2, but it has been reported that some plasmid preparations transfect at this ratio.See section 2.6, Optimization of FuGENE® HD Transfection Reagent:DNA ratio, for more information andvisit /fugene/hdFuGENE® HD Trans-fection Reagent wasaliquotedCheck that FuGENE® HD Transfection Reagent is stored in the original container. If the reagent wasaliquoted into plastic containers, there is a high chance of inactivation. Make sure the reagent isimmediately mixed with the dilute DNA either by vortexing or pipetting up to 10 – 15 times.FuGENE® HD Trans-fection Reagent cameinto contact withplastic or wasinadequately mixedRepeat transfection, carefully pipetting FuGENE® HD Transfection Reagent directly into the serum-freemedium, being careful not to touch the sides of the container while adding the FuGENE® HD Transfec-tion Reagent to the diluted DNA. If the FuGENE® HD Transfection Reagent is added too gently, it maylayer on top of the medium, thus making contact with the plastic.Transfection complexwas formed in serum-containing mediumCheck original bottle of medium used for complex formation. Repeat experiment using new bottle ofOpti-MEM that does not contain any additives (e.g., serum, antibiotics, growth enhancers, heparin,dextran sulfate, etc.). Try forming the complex in sterile water or plain DMEM.Media and mediacomponentsDifferent media and media components may influence the level of transfection efficiency andsubsequent growth of the transfected cells, as well as expression of the recombinant protein. Some lotsof sera have been reported to interfere with optimal transfection.Quality and/or lot-to-lot differences that affect transfection experiments have been noted in both seraand media. Check that the medium and/or serum is from the same lot that worked previously. Try newlots or a different vendor.Culture may becontaminated withmycoplasmaCultures contaminated with mycoplasma have been shown to have decreased transfection efficacy.Determine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Inconsistent results Ratio or amount oftransfection complexis at the edge ofperformance plateauInitial experiments should be completed to determine the ratios, amount of complex to be added, andlength of time for complex formation for optimal performance. In our experience, we have found the pla-teau to be relatively broad. We recommend that future experiments be performed with ratios, incubationtime, and amounts of complex that were in the middle of the plateau. If conditions are selected at theedge of the plateau, very small procedural differences may cause large differences in the resulting pro-tein expression. Increased consistency may be achieved by shifting parameters away from the edge ofthe plateau to the middle of the plateau.Transfection complexformation:timing,amounts, and ratioFormation of the complex involves a multifaceted interaction between the transfection reagent and DNAas well as biological parameters. Differences in any of the components or techniques may result ininconsistencies. If results do not meet your expectations, then repeat the optimization experimentselecting areas near the plateau found in previous experiments. For current experiments, determine ifyou should use a different ratio, length of time, or amount of complex for more consistent transfectionresults.Extensive testing of the FuGENE® HD Transfection Reagent is performed on two cell lines: one easy totransfect and one very difficult to transfect. All reagent lots must pass this rigorous testing before wemake it available to you. However, we cannot test all cell lines, media, sera, and vectors; in your labora-tory, you may find slight differences in the optimal ratio, amount of complex, or time for complex forma-tion for some lots of FuGENE® HD Transfection Reagent.Cells For consistent results, cells must be properly maintained. Cells change with passage level, passage conditions, media, and sera. For some cell lines, these changes have little to no effect on transfectionexperiments, but for other cell lines, these changes have profound effects. Each cell type may have a dif-ferent optimal transfection condition. Optimal values for a single cell type may also change slightly withvector construct and type of protein expressed.Observation Possible cause RecommendationSigns of cytotoxicity Transfected protein iscytotoxic or isproduced at highlevelsReduced viability or slow growth rates may be the result of high levels of protein expression, as the cell’smetabolic resources are directed toward production of the heterologous protein. The expressed proteinmay also be toxic to the cell at the level expressed.To analyze cytotoxicity, prepare experimental controls as described below.Prepare extra control wells containing:ቢ Cells that are not transfectedባ Cells treated with DNA alone (e.g., without FuGENE® HD Transfection Reagent)ቤ Cells treated with FuGENE® HD Transfection Reagent alone (no DNA added)ብ Cells transfected with a non-toxic or secreted protein.Compare experimental transfected cells to cells in the control wells (described above). Considerrepeating the experiment with a secreted reporter gene such as SEAP, hGH, or a standard ␤-gal controlvector. Cells expressing SEAP should show little to no evidence of cytotoxicity.Too much transfec-tion complex fornumber of cellsIncrease the number of cells plated, and/or decrease the total amount of complex added to the cells. Trydifferent ratios and allow the complexes to form for different time intervals. Add different amounts ofcomplex; for example, make the complex as usual but add 75%, 50%, or 25% of the usual amounts toeach well. See Suboptimal FuGENE® HD:DNA Ratio in "Low transfection efficiency" section of this tablefor details or optimization protocol.Culture may becontaminated withmycoplasmaDetermine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Cells may not behealthyAssess physiological state of cells and the incubation conditions (e.g., check incubator CO2, humidity,and temperature levels). Observe cells prior to each passage for morphology and absence of contami-nants. Make sure cells do not overgrow. Routinely passage cells prior to reaching confluency. Make surethat culture media and additives are within expiration date and have been stored properly.Diluent is toxic to thecellsDMEM is toxic to some insect cell lines. For these cells, prepare the transfection complex in sterilewater. You may also try forming the complex in the medium in which the cells are growing, providingthat the medium does not contain serum, heparin, or dextran sulfate.Plasmid preparationcontaminated withendotoxinEndotoxin is reported to be cytotoxic to sensitive cell lines.If above tests provenegative, FuGENE®HD TransfectionReagent may not beoptimal for your cells.Try FuGENE® 6 Transfection Reagent*, DOTAP Liposomal Transfection Reagent*, DOSPER LiposomalTransfection Reagent*, or X-tremeGENE Q2 Transfection Reagent*.High protein-expression levelsHigh expression levels of certain intracellular proteins (e.g., Green Fluorescent Protein [GFP]) may becytotoxic to some cell types. Cell proliferation, toxicity, and cell death may be monitored using Apoptosisand Cell Proliferation products from Roche Applied Science (visit /apoptosis for more information).Media and mediacomponentsTest different media and optimize the level of each medium component for these cytotoxic effects.Although it is not usually necessary to remove the transfection complex following the transfection step,it may be necessary to feed your cells with fresh media for extended growth periods. This is particularlyimportant if the transfected cells are allowed to continue to grow for 3 – 7 days to provide maximalprotein expression.。

FuGENE 转染流程

FuGENE HD真核细胞转染流程FuGENE HD Transfection Reagent Quick Protocol.pdfFuGENE HD Transfection Reagent.pdf1.细胞和质粒的准备①将约5-10×105A549细胞接种于6孔细胞板中，用含10%小牛血清的F-12k培养过夜，约60%~80%铺满时用于转染。

注：一般荧光试验细胞稀一点为宜；WB试验细胞密一点为宜②转染时所用质粒均用转染专用质粒抽提试剂盒无菌提取，抽提后测定其浓度和纯度，浓度在0.1μg/μL以上且纯度在1.8±0.5 (OD260/OD280)的质粒用于转染，质粒提取的具体步骤按说明书提供的方法进行。

（注：质粒浓度测定未必可行，需同时进行酶切和PCR鉴定方可。

）③按照转染剂量，计算质粒使用浓度2.转染流程注：由于不同转染试剂对质粒大小、性质、转染量、细胞的种类有很强的特异性，必须谨慎选择转染试剂。

以多次转染成功使用的转染试剂为佳。

①将培养板中的上清弃去，用细胞用无抗无血清F12K洗涤三次后，每孔加入800ul 无抗无血清F12K。

②取出FuGENE HD转染试剂，使其温度上升到室温。

③计算好质粒、转染试剂、以及无抗无血清F12K的量，最终液体总量为100 ul。

准备好无菌指形管，按计算结果加入90-98ul的无抗无血清F12K，加入2ug 质粒，振荡器混匀；随后加入6ul FuGENE HD转染试剂，立即震荡混匀（转染试剂加入时不能沾到管壁上！）④室温静置7-12分钟后，将质粒：转染试剂混合液均匀滴加在细胞平板孔中，吹打混匀或者震荡混匀。

放入37℃培养箱孵育。

⑤转染后2 h后加入含有血清的F12K，使血清终浓度达到1-4%，上清总量达到1.5-2 ml。

细胞平板放入37℃培养箱培养24-48h。

⑥转染后特定时间点，用4℃预冷的PBS漂洗细胞3次，加入含有PMSF的Western细胞裂解液冰上裂解15min。

一般转染操作步骤

一般转染操作步骤
1、稀释转染试剂
将6ul转染试剂FuGENE加到装有90-98ul预热到室温培养基的无菌聚丙乙烯管中，立即混匀，室温放置5min。

注：勿将未稀释的转染试剂FuGENE碰到管壁，造成稀释不充分。

2、制备转染物
加2ug的质粒DNA至稀释的转染试剂中，立即混匀，室温放置15min。

注：勿超过45min，否则影响转染效率。

3、添加转染物至细胞培养液
将2-10ul的转染物加到含有100ul细胞培养液的96孔板中，轻轻吸打或者左右摇晃10-30S以充分混匀。

推荐：5ul作为起始点
4、孵育培养
置于CO2培养箱中，培养24-48h检测。

转染试剂FuGENE：DNA=3:1
质粒DNA浓度：0.2-1 ug/ul
96孔板每孔2-10ul转染物成分：0.15-0.6ul的转染试剂+0.04-0.2ug的质粒DNA。

转染步骤范文

转染步骤范文转染是指将外源DNA导入到受体细胞中的过程，用于基因工程、基因治疗等研究和应用。

转染步骤是实现转染的关键步骤，包括细胞培养、转染试剂制备、转染试剂处理、细胞培养和分析。

以下是转染步骤的详细描述。

第一步：细胞培养在进行转染实验之前，首先需要培养并扩增所使用的细胞。

细胞的培养基因常见的有DMEM、RPMI等，培养基中会添加适当的酵素抑制剂（如胰酶）和生长因子，以促进细胞的生长和增殖。

第二步：转染试剂制备转染试剂可以是化学试剂，也可以是病毒载体或质粒DNA。

化学试剂常见的有聚乙烯亚胺（PEI）、脂质体、阳离子聚合物等。

病毒载体包括腺病毒、逆转录病毒、腺相关病毒、大肠杆菌之Coli phage等。

质粒DNA则是将外源DNA经过酶切和纯化处理得到的。

第三步：转染试剂处理在这一步中，将转染试剂与细胞一起孵育，以使转染试剂与细胞内部的DNA互相结合。

转染试剂的使用方法根据具体试剂的特点而定，常见的方法有磷酸钙共沉淀、脂质体包裹、病毒载体包装等。

第四步：细胞培养和分析在细胞孵育一定时间后，可以对转染效率进行分析。

常见的分析方法包括荧光显微镜观察、流式细胞术、PCR等。

通过这些分析方法，可以观察到转染后的外源DNA是否成功导入到细胞，并根据实验需要进行下一步操作。

除了以上步骤，还有一些实验时需要注意的要点，例如选择合适的细胞密度和培养时间、优化转染条件、进行有效杀菌等。

同时，也需要仔细阅读相关实验操作手册，并参考先前类似研究的文献，以确保实验的准确性和可重复性。

总结起来，转染步骤是进行基因转染的重要环节，包括细胞培养、转染试剂制备、转染试剂处理、细胞培养和分析。

通过精确执行每个步骤，并结合适当的方法和实验操作要点，可以高效地将外源DNA导入到受体细胞中，实现基因工程和基因治疗等研究和应用的目的。

转染试剂的中文详解

各种转染试剂的中文转染方法2014-05-06丁香园FuGENE6（Roche）转染步骤：转染前一天将细胞分至培养板，转染当天细胞应50-80％融合。

将细胞以1-3×105/2 ml接种于6孔板后孵育过夜将达到如此密度。

将FuGENE6 Reagent在室温静置10-15分钟以平衡到室温。

使用之前将FuGENE6颠倒混匀一下。

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

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

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

4.室温孵育20分钟。

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

6.将转染复合物加入细胞，混匀使之均匀分布。

7.3-8小时后，加入血清或换成含血清的营养液。

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

细胞铺板在2 ml含血清，不含抗生素的正常生长的培养基中。

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

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

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

NOTE：若使用DMEM培养基，则需在5分钟内同稀释的DNA混合。

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

室温放置20分钟。

5.（optional）将6孔板中的旧营养液吸出，用无血清培养基清洗两次。

转染步骤及经验(精华)

转染步骤及经验（精华）一、基础理论转染是将外源性基因导入细胞内的一种专门技术。

分类：物理介导方法：电穿孔法、显微注射和基因枪；化学介导方法：如经典的磷酸钙共沉淀法、脂质体转染方法、和多种阳离子物质介导的技术；生物介导方法：有较为原始的原生质体转染，和现在比较多见的各种病毒介导的转染技术。

理想细胞转染方法，应该具有转染效率高、细胞毒性小等优点。

病毒介导的转染技术，是目前转染效率最高的方法，同时具有细胞毒性很低的优势。

但是，病毒转染方法的准备程序复杂，常常对细胞类型有很强的选择性，在一般实验室中很难普及。

其它物理和化学介导的转染方法，则各有其特点。

需要指出的一点，无论采用哪种转染技术，要获得最优的转染结果，可能都需要对转染条件进行优化。

影响转染效率的因素很多，从细胞类型、细胞培养条件和细胞生长状态到转染方法的操作细节（见后文）。

二、转染操作流程（以常用的6孔板为例）(1) 细胞培养：取6孔培养板，以3x104/cm2密度铺板，37℃5%CO2培养箱中培养至70%～90%汇合。

(不同细胞略有不同，根据实验室优化的条件进行，汇合过分，转染后不利筛选细胞)。

(2) 转染液制备：在EP管中制备以下两液(为转染每一个孔细胞所用的量)A液：用不含血清培养基稀释1-10μg DNA，终量100μL，B液：用不含血清培养基稀释对应量的转染试剂，终量100μL；轻轻混合A、B液（1:1混匀），室温中置15分钟，稍后会出现微浊现象，但并不妨碍转染。

(3) 转染准备：用2mL不含血清培养液漂洗两次，再加入2mL不含血清及PS的培养液。

(4) 转染：把A/B复合物缓缓加入培养液中（缓慢滴加），轻轻摇匀，37℃温箱置6～8小时，吸除无血清转染液，换入正常培养液继续培养。

三、转染注意事项1. 血清A. DNA-阳离子脂质体复合物形成时不能含血清，因为血清会影响复合物的形成。

B．一般细胞对无血清培养可以耐受几个小时没问题，转染用的培养液可以含血清也可以不加，但血清一度曾被认为会降低转染效率，转染培养基中加入血清需要对条件进行优化。

转染步骤及经验

转染步骤及经验
1.根据要进行体细胞转染的实验的特殊要求，选择适当的载体质粒，以及目标细胞。

2.制备转染的DNA样本：根据质粒的大小和实验的要求，严格控制样本的DNA浓度，并确保其质量良好，无杂质；
3.准备转染液：将转染的DNA样本和必要的添加剂放入适量的
PBS/DMEM中混合，形成转染液；
4.细胞放入转染液中：把细胞放入适量转染液中，使细胞与转染液充分混合；
5.细胞转染：细胞转染的方法有优化的转染技术（如脉冲转染）和非优化转染技术（如胞浆转染），根据实验对转染效果的要求，选择合适的转染技术；
6.细胞休眠：细胞休眠后，为后续实验提供了理想的条件，简化接下来的操作。

7.细胞筛选：有些载体质粒将特定的标记物（如GFP）植入细胞内，接着，使用不同颜色的染料或者发光技术，筛选出转染后的细胞，这将有助于后续的实验。

8.检测转染效率：使用细胞膜染色，PCR，蛋白表达分析来检测转染效率，以证实转染是否成功。

9.细胞接种：转染细胞分离后，接种到HMVEC或其他细胞上，以复制体系，以便进一步的研究。

细胞转染是一个复杂的过程，需要仔细地操作。

转染步骤及经验

转染步骤及经验转染是在生物实验中将外源基因导入到细胞中的常用操作步骤之一。

本文将介绍转染的步骤及经验，并提供一些实用的技巧，旨在帮助读者顺利完成转染实验。

一、实验准备在进行转染实验前，必须做好充分的实验准备工作。

以下是一些关键准备步骤：1.1 细胞培养与处理- 培养适量的目标细胞，并确保其良好的生长状态。

- 处理细胞，使其处于适宜转染的状态。

例如，对于贴壁细胞，可以使用酶消化将细胞分散为单个细胞。

1.2 载体准备- 准备带有目标基因的载体。

这可以是质粒、病毒等。

- 使用合适的方法制备高质量的载体DNA或RNA。

1.3 转染试剂- 准备合适的转染试剂，如转染剂、离子可溶液等。

不同类型的细胞需要使用不同的试剂。

二、转染步骤转染步骤的具体操作根据不同的实验目的和细胞类型可能会有所不同。

以下是一般的转染步骤：2.1 细胞处理- 将要转染的细胞洗涤并收集到离心管中。

- 注意不要创建过高的转染细胞密度，以免影响转染效率。

2.2 与载体混合- 将载体与转染试剂混合，形成载体-试剂复合物。

根据实验需要可做不同浓度的复合物。

- 注意操作过程中避免产生气泡，以防止对细胞的损伤。

2.3 加入转染复合物- 缓慢地将转染复合物滴加到细胞中，确保复合物均匀分布在细胞表面。

- 转染时间和温度应根据细胞类型和所用试剂进行优化。

2.4 培养细胞- 按照细胞所需的培养条件将细胞孵育在恰当的培养基中。

- 在培养期间，根据实验设计进行后续处理，如观察表型、提取蛋白等。

三、经验分享3.1 优化试剂浓度- 不同的细胞系对试剂的敏感度不同，需要根据实验经验进行优化。

试验应根据需要尝试不同浓度的试剂。

3.2 选择合适的转染方法- 根据研究目的和所用试剂的特性，选择合适的转染方法。

常见的转染方法包括化学法、电穿孔法、病毒载体法等。

3.3 控制转染时间和温度- 不同细胞对转染复合物的吸收速度有差异，应该根据细胞类型和试剂特性确定合适的转染时间和温度。

转染操作步骤

转染操作步骤嘿，朋友们！今天咱来聊聊转染操作步骤这档子事儿。

转染啊，就好比是一场细胞的奇妙冒险！想象一下，你要把一些外来的东西，比如说核酸啦，送进细胞这个小小的“城堡”里。

这可不是一件容易的事儿，但别怕，跟着我一步一步来，保证能让你搞定它。

首先呢，得准备好你的“道具”。

细胞得养好啦，状态得杠杠的，就像要出征的战士一样精神。

还有那些转染试剂，可别小瞧它们，它们可是这场冒险的重要“伙伴”。

然后呢，就是关键的时刻啦！把核酸和转染试剂按照一定的比例混合起来，这就像是给它们牵红线，让它们“相亲相爱”。

搅拌均匀咯，可别马虎。

接下来，把这混合好的“魔法药水”慢慢滴加到细胞里。

嘿，这可得小心点，别跟细胞闹别扭。

滴加的时候，就像是给细胞送上一份神秘的礼物，得轻拿轻放。

之后呢，就是等待啦。

让细胞和这些外来的东西好好相处，给它们时间互相熟悉熟悉。

这时候你可别闲着，时不时去观察观察，看看有没有什么奇妙的变化。

过了一段时间，你就可以去看看成果啦！看看那些核酸是不是成功地住进了细胞这个“城堡”里。

要是一切顺利，哇塞，那可太棒啦！就好像你成功地完成了一项了不起的任务。

哎呀，这转染操作说起来简单，做起来可得细心再细心。

就像走钢丝一样，一步都不能错。

要是不小心出了岔子，那可就前功尽弃啦。

不过别担心，多练几次，你肯定能掌握其中的窍门。

转染这事儿啊，真的很神奇。

它能让我们把想要的东西送进细胞里，让细胞发生一些奇妙的变化。

这就像是给细胞施了魔法一样，是不是很有意思？所以啊，朋友们，大胆去尝试吧，别怕失败，失败是成功之母嘛！只要你有耐心，有细心，转染操作绝对难不倒你！加油哦！。

细胞转染流程

细胞转染流程细胞转染就像是给细胞送一份特殊的“快递”，这个“快递”里装着我们想要细胞接受的东西，比如说新的基因之类的。

那咱们就开始这细胞转染之旅吧。

在做细胞转染之前，你得先把细胞照顾好。

细胞就像娇嫩的小宝贝，要给它们一个舒适的“家”，这个“家”就是培养皿啦。

得把细胞在合适的培养基里养着，温度要刚刚好，就像人住在温度适宜的房子里一样舒服。

要是温度不合适，细胞就会闹脾气，就像人在过热或者过冷的环境里会不舒服一样。

而且培养基里的营养成分得充足，就好比给孩子的饭菜得营养全面，这样细胞才能长得壮壮的，有精力来接受我们要给它的“快递”。

接下来就是准备转染试剂和要转染的核酸啦。

这转染试剂就像是专门的快递员，它的任务就是把核酸这个“包裹”送到细胞里面去。

核酸呢，可以是DNA，也可以是RNA，这就看你想让细胞做什么了。

你得按照说明书，精确地量取转染试剂和核酸的量。

这就好比做菜的时候，盐放多了或者放少了都不行，得恰到好处。

要是转染试剂或者核酸的量不对，那这个“快递”可能就送不好，细胞可能就接收不到正确的“包裹”。

然后就是把转染试剂和核酸混合在一起。

这时候就像是快递员把包裹打包好一样，要轻柔地操作。

不能太粗暴，不然可能会把“包裹”弄破或者弄乱了。

混合好之后，还得让它们静静地待一会儿，就像给快递员一点时间整理一下包裹，确保一切都准备妥当。

再把这个混合好的东西加到细胞培养皿里。

这就像是把打包好的快递送到细胞的“家门口”了。

细胞看到这个“快递”，就会想办法把它“拿”进去。

这个过程有点像细胞在门口迎接快递员，然后把包裹拿进自己的家里。

不过细胞拿这个“包裹”进家可不容易，有时候可能会失败，就像快递员有时候会送错地址一样。

在细胞接收这个“快递”的过程中，我们得在旁边静静地看着。

就像等着孩子拆礼物一样，充满期待又有点小紧张。

我们得观察细胞的状态，看看它们有没有什么不良反应。

如果细胞开始变得无精打采，或者形态变得奇怪，那就可能是转染出问题了。

原代细胞转染步骤

原代细胞转染步骤原代细胞转染是一种将DNA、RNA或蛋白质等生物分子导入细胞内的方法，它被广泛应用于细胞遗传和药物开发等领域。

在进行原代细胞转染时，需要仔细的操作并遵循一系列的步骤。

首先，我们需要准备好原代细胞和转染试剂。

原代细胞应当在生长期且处于状态好的状态，如要进行转染的细胞来自动物体内，则需要通过适当的方法进行分离和培养。

转染试剂可以是多种选择，例如：化学试剂、聚合物或与病毒相关的转染试剂。

在选择试剂时，需要考虑到细胞种类和所需转移物之间的互补性和相容性。

接下来，我们需要确定转染试剂的浓度。

过高的浓度可能会导致细胞死亡，而过低的浓度则会降低转染的效果。

因此，应该先进行不同浓度的试验，最后选择最适合的浓度。

然后，我们需要按照试剂提供的说明书或参考文献的指导操作。

通常试剂需要预先混合，试剂的添加需要分几步进行，并在每一步后轻轻的混合。

此外，由于细胞对试剂的反应不同，因此我们需要在不同的细胞类型中进行试验，以找到最佳的处理方式。

接下来，我们需要将转染试剂与细胞混合。

这需要轻轻拍打培养皿或使用转染通道将混合物加入细胞培养基中。

随着细胞被转染并进入生长周期，原代细胞从之前的瞬间转化为可以被恒定表达的细胞。

最后，在细胞与试剂混合后，我们必须为其提供适当的环境以促进细胞生长。

这通常意味着需要为细胞提供充足的营养和条件，例如适当的CO2浓度和恰当的温度。

在细胞转染过程完成后，需要进行逐步筛选和鉴定以评估转染的效果和细胞生长的状态。

总之，虽然原代细胞转染是一个复杂的过程，但结合我们了解的流程和注意事项进行，不仅能使转染过程容易和高效，同时也能提高转染的成功率，使我们更快速的满足实验的要求。

FuGENE 6 转染步骤

配方和包装：FuGENE® 6是由脂质与其他组分按照合适比例混合，溶于80%乙醇而成。

试剂通过0.1μm的滤器过滤并分装至小玻璃瓶。

注意事项：转染前须保证FuGENE®6温度已经达到室温，使用前须上下颠倒数次进行简单混匀。

建议使用标准的24孔板作为FuGENE® 6转染试剂的支架。

请不要将原瓶中的FuGENE®6进行分装。

尽可能减少FuGENE®6原液与塑料制品的表面接触。

不要使用硅化的枪头或离心管。

在稀释FuGENE® 6时，请务必保证将FuGENE® 6直接混入培养基，而不要接触到离心管。

（研小弟注：还要防挥发，毕竟溶于80%乙醇，所以也不宜分装）抗生素的使用：在细胞系的常规培养时可以使用抗生素。

但在转染时抗生素的存在可能会影响转染的效率以及转染细胞的整体健康。

除非之前已经在转染细胞中测试过，否则我们不推荐在转染培养液中添加抗生素。

转染步骤：以下是一个简易的中文转染步骤示意图具体的转染步骤包括：•细胞的接种在转染前一天接种细胞，保证在转染时细胞的汇合度在50-80%，悬浮细胞可以在转染当天接种。

通常96孔板中每个孔适宜接种100μl含1-2×104的贴壁细胞或2×104到1×105的悬浮细胞。

对于其他规格的培养板的推荐细胞数，可参考下表（数据针对Corning公司生产的培养板）。

96-well 0.32 1×24-well 1.88 5×12-well 3.83 10×6-well 9.4 30×35mm 8 25×60mm 21 65×100mm 55 170×•FuGENE® 6转染试剂的准备1.转染前保证FuGENE® 6温度已经达到室温2.使用前上下颠倒数次进行简单混匀。

•常规转染步骤我们强烈推荐您针对每个细胞系建立优化转染条件。

FuGENE6转染试剂中文操作说明

FuGENE6转染试剂中文操作说明FuGENE®6原是Roche公司的旗舰转染产品，如今已归为Promega公司旗下。

它是一种非脂质体转染试剂，广泛适用于高效转染各种细胞系，且细胞毒性非常低。

由于使用该转染试剂转染时不要求去除血清或培养液，并且转染后也不要求换液去除，因而使用起来非常方便。

配方和包装：FuGENE® 6是由脂质与其他组分按照合适比例混合，溶于80%乙醇而成。

试剂通过0.1μm的滤器过滤并分装至小玻璃瓶。

注意事项：转染前须保证FuGENE® 6温度已经达到室温，使用前须上下颠倒数次进行简单混匀。

建议使用标准的24孔板作为FuGENE®6转染试剂的支架。

请不要将原瓶中的FuGENE®6进行分装。

尽可能减少FuGENE®6原液与塑料制品的表面接触。

不要使用硅化的枪头或离心管。

在稀释FuGENE® 6时，请务必保证将FuGENE® 6直接混入培养基，而不要接触到离心管。

（研小弟注：还要防挥发，毕竟溶于80%乙醇，所以也不宜分装）抗生素的使用：在细胞系的常规培养时可以使用抗生素。

但在转染时抗生素的存在可能会影响转染的效率以及转染细胞的整体健康。

除非之前已经在转染细胞中测试过，否则我们不推荐在转染培养液中添加抗生素。

通常96孔板中每个孔适宜接种100μl 含1-2×104的贴壁细胞或2×104到1×105的悬浮细胞。

对于其他规格的培养板的推荐细胞数，可参考下表（数据针对Corning公司生产的培养板）。

FuGene HD试剂转染GFP入A549细胞关键条件的优化

FuGene HD试剂转染GFP入A549细胞关键条件的优化时文艳;赵良中;张铎;李妍【摘要】目的优化FuGene HD试剂转染外源基因入A549细胞的实验条件.方法培养A549细胞,改变FuGene HD试剂/质粒DNA比例以及转染GFP基因的时间,荧光显微镜观察和计数绿色荧光蛋白表达细胞以非表达细胞,并计算转染效率和细胞活力.结果 FuGene HD试剂(μL)/质粒DNA(μg)比例为2.5,转染时间12 h,转染效率为(84.6±5.2)%;而细胞活力为(94.8±4.7)%.结论 FuGene HD试剂/质粒DNA比例为2.5,而转染时间为12 h,可在A549细胞获得较高转染效率和细胞活力.【期刊名称】《吉林医药学院学报》【年(卷),期】2010(031)006【总页数】2页(P311-312)【关键词】基因;转染效率;细胞活力【作者】时文艳;赵良中;张铎;李妍【作者单位】吉林医药学院病原学教研室,吉林,吉林,132013;吉林医药学院病原学教研室,吉林,吉林,132013;吉林医药学院病原学教研室,吉林,吉林,132013;吉林医药学院病原学教研室,吉林,吉林,132013【正文语种】中文【中图分类】R346将外源基因转入靶细胞是免疫学、细胞生物学和遗传学研究的基本技术[1]。

A549细胞为贴壁生长的肺癌细胞，在其贴壁生长状态下，可采用病毒介导转染、磷酸钙转染、脂质体以及非脂质体脂类试剂转染等方法导入外源基因。

新型非脂质体试剂FuGene HD可将大片段DNA转染入多种类型细胞，但需要调整转染试剂与DNA比例和转染时间，来优化转染条件。

本研究通过筛选转染FuGene HD和绿色荧光蛋白质粒DNA的比例以及转染时间，来优化转染A549细胞的条件。

1.1 材料A549细胞源自ATCC，由本实验室购买并保存。

pEGFP-N1质粒由本实验室保存，纯化pEGFP-N1 DNA溶于去离子水(1 mg/mL),并置-20 ℃保存。

FuGENE HD 转染试剂简明操作指导说明书

FuGENE®HD转对于表格中未列出的细胞，采用以下步骤进行实验后，可寻找到适用于您所研究细胞的最佳质粒与转染试剂的配比：FuGENE®HD 转染试剂简明操作步骤II. 准备实验所需的细胞、试剂及耗材：•细胞：在合适的培养条件下（建议采用无抗生素培养基，可以含任意比例的血清），实验前细胞系培养至长满60%-80%，原代细胞培养至适当的时间；•转染试剂：使用前，将转染试剂放至室温，颠倒混匀(FuGENE®HD转染试剂储存于4℃，如不慎将FuGENE®HD冰冻，融化后可能会看到不溶物。

这时可将试剂短暂升温至37℃，颠倒混匀后不溶物消失)；•质粒：携带报告基因或荧光蛋白的质粒，用于计算转染效率;•无菌、无血清培养基：用于配制质粒与转染试剂的混合物；•无菌的枪头、离心管，超净工作台等。

简易流程图培养细胞至60%-80%融合度配制质粒与转染试剂的混合物混合物加入培养的细胞中检测，计算转染效率I I I.操作步骤1.将无血清培养基预热至室温，按下表的比例配制质粒与转染试剂的混合物：FuGENE®HD与质粒DNA的比例4:1 3.5:13:1 2.5:12:1 1.5:1培养基100μl100μl100μl100μl100μl100μl质粒2μg2μg2μg2μg2μg2μg FuGENE®HD8μl7μl6μl5μl4μl3μl注意：FuGENE®HD应直接加入培养基中，不用沾到离心管的管壁上2.混合物室温静置10-15 min。

3.将混合物滴加至培养的细胞中，96孔板每孔加入5µl，其他孔板的加入量可以按照右侧的表格进行计算。

摇晃或吹打混匀。

继续培养24-48 hr。

4.检测转染效率。

进行报告基因检测或计数表达绿色荧光蛋白的细胞数目，确定最佳的质粒与转染试剂的比例。

注：FuGENE®HD转染试剂对细胞几乎没有毒性，遇到特别难转染的细胞，希望提高转染效率时，可以将混合物的加入量加倍至10μl/孔或15μl/孔（96孔板，其他培养板按培养面积放大）。

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