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. Successfully transfected cell types include:. 7 Cells with Lipofectamine 2000 AfCS Procedure Protocol PP00000182 Page 2 Preparation of RAW 264. 5–5 微量离心管制备:10分钟 孵育:5分钟 最终孵育:1–3天 Lipofectamine 试剂在网站上查看相关产品 3000试剂采用了专利配方,可将核酸转染至各种真核细胞中,尤其是难以转染的. " Review date: 03 Feb 2017 | Invitrogen™ Lipofectamine® 3000 Transfection Reagent. Lipofectamine® 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 000 cells/well) 90% confluency at the day of transfection (100% at the day of analysis) 53 ng and 82 ng of MC are equimolar to 82 ng and 126 ng of pCMV, respectively. Life Technologies is a global biotechnology tools company dedicated to improving the human condition. TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent MCF10A Breast cancer cells Complete growth medium Component Cat. 当前位置: 文档下载 > 所有分类 > 自然科学 > 生物学 > Life Technologies Lipofectamine 3000 中文说明 免费下载此文档 侵权投诉 Life Technologies Lipofectamine 3000 中文说明. Lipofectamine ™ 2000 는 DNA 와 RNA 모두를 진핵 세포로 전달할 때 사용하는 제품입니다. 7 cells, growing in RAWGM1, prior to transfection. transfection reagent Lipofectamine® 3000. The lipofectamine 2000 protocol suggests a maximum of 800ng plasmid DNA for each well in a 24-well plate. Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. 7ug of appropriate viral plasmid. Especially Lipofectamine 3000, as it is a most efficient versatile reagent for the widest range of cell types including d…. However, they can be difficult to produce. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Transfection by Lipofectamine (using Invitrogen LipofectamineTM) 1. lipofectamine 2000 vs 3000. Lipofectamine® 2000 DNA Transfection Reagent Protocol See page 2 to view a typical DNA transfection procedure. 11668-019 Size: 1. Transfection of cells with plasmid DNA is well optimized, and the procedure is simple and quick. Lipofectamine® 2000 and Lipofectamine® 3000 The transfection performance of cell-specific DNA-In™ reagents were compared to those of Lipofectamine® 2000 and Lipofectamine® 3000 (LF3K). If there appears to be large-scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates. Standard versus Reverse Transfection. Plate cells so they will be 70-90% confluent at the time of transfection. 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. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. i am going to transfect my 293 cells for the production of Adenovirus so i would like to know your opinions about it. LipofectamineŽ 2000 Cat. the total volume and photons outside the detection volume are not detected. This video shows you how to pronounce Lipofectamine. General Note: Plate cells for 2-12hrs (but not over 24 hours) prior to transfection to achieve a 40-80% confluence at the time of transfection. Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). Lipofectamine 2000 Transfection Reagent is best transfection reagent available from Life Technologies for the co-transfection of siRNA and plasmid DNA. Bioz Stars score: 99/100, based on 725 PubMed citations. 58056: Effectene 1 ml 301425: kit: Qiagen: 58054: Lipofectamine 2000 0. TRANSIENT TRANSFECTION OF MAMMALIAN CELLS WITH LipofectAMINE. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Incubate 5 min at room temp then mix lipofectamine and siRNA dilutions. - Characterization of transfection efficiency of IMR32 through calcium phosphate and lipofectamine 3000 transfection. Lipofectamine® 2000 and Lipofectamine® 3000 The transfection performance of cell-specific DNA-In™ reagents were compared to those of Lipofectamine® 2000 and Lipofectamine® 3000 (LF3K). I did get the knockdown to work when using my siRNA with lipofectamine 2000, however. Liposome-based DNA carriers may induce cellular stress response Figure 1: pin. It increases the likelihood of successful cleavage and recombination with TALENs or CRISPRs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. The Mechanism of Lipofectamine 2000 Mediated Transmembrane Gene Delivery Shaohui Cui, Shubiao Zhang, Huiying Chen, Bing Wang, Yinan Zhao, Defu Zhi. Lipofectamine® 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines; more than ten-fold. Lipofectamine® 3000 Transfection Reagent Lipofectamine ® 3000 reagent leverages our most advanced. Lipofectamine® 3000 is a transfection reagent that was developed to improve transfection efficiency in many difficult to transfect cell types. 02% transfection efficacy, similar to the group of Lipofectamine 3000 & P3000 treatment, the treatment of Lipofectamine 3000 alone recorded low transfection efficacy in most cases, with minor transfection efficacy recorded in HL60 (2. This is the first time I will have used Lipofectamine 3000, previously I only used Lipofectamine 2000, and I am wondering if anyone has found an optimal Lipo 3000 / DNA ratio for this cell line. with that of Lipofectamine 3000 and Lipofectamine 2000. We have had success using Lipofectamine reagents in the past and the Lipofectamine 3000 appears to be the best option. There's a difference between the two protocols - lipofectamine 3000 has an extra reagent that needs to be added. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hard-to-transfect and common cells (e. If there appears to be large-scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates. " Review date: 03 Feb 2017 | Invitrogen™ Lipofectamine® 3000 Transfection Reagent. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. I was recently having issues using lipofectamine 3000 so did a lot of troubleshooting. 3-fold in. Lipofectamine 3000 and P3000™ Enhancer Reagent (Thermo Fisher Scientific Inc) were used for transient plasmid transfections according to manufacturer's protocol and the cells were cultured for 24 h. Lipofectamine® 3000 Transfection,描述 Lipofectamine® 3000转染试剂中包含了我们最为先进的脂质体纳米颗粒技术,能够提供卓越的转染性能,改善相关应用的结果及可重复性。此试剂具有优异的转染效率,并可提高细胞活性,广泛适用于难转染的及常. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. Studies were undertaken to evaluate and compare the transfection efficacy. 75ml 11668027: tube: Life Technologies: 58075: Lipofectamine 2000 1. I've had great results with my normal cell lines, my GBM ones are a bit more frustrating but they are inherently harder to transfect. 11668-019 Size: 1. Despite this, a satisfactory mechanism-based explanation of their. 5 % FBS to the transfection complexes, once DNA and lipofectamine is mixed and incubated. Co-transfection of multiple plasmid DNAs is a technique increasingly employed by biology researchers. In this study, we demonstrated that using an improved transfection reagent, such as Lipofectamine® 3000, helps to eliminated many of the pain points. Enhanced protein expression using Lipofectamine. 3000 (GP3K), we demonstrate a significant increase in transfection of RAW 264. Lipofectamine™ 2000 Reagent should not be frozen. A protocol for Hek293 would be a bonus. 75 µL)。 在新型基因组编辑的相关应用中获得高转染效率. A growing body of evidence suggests that stem cell-derived exosomal microRNAs (miRNAs) could be a promising cardioprotective therapy in the context of hypoxic conditions. Protocol for Lipofectamine Transfection in NIH-3T3 Cells Using Cells Plated in 8-well Plates 1) Mix 0. 25 ml of Lipofectamine RNAiMAX diluted 33. Transfection of PC12 Cells with Lipofectamine. 求lipofectamine 2000中文说明书 ----英文的我有了,看不动! 回答:" T:" 不同的细胞已及转不同面积的孔板操作起来略有不同,以转染六孔板的贴壁. Transfection of NIH3T3 cells, Hela, Swis 3T3, 293T with Lipofectamine 2000 1. We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. Load equal amounts of protein (20 μg) into the wells of a mini (8. We describe this new technology and how this simple versatile reagent delivers efficient, reporducible transfection of both DNA and RNA ino a wide range of hard-to-transfect, biologically relevant cell models. If there appears to be large-scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates. Each tube gets 8mls of OPTI-MEM, 32ug of gag pol, 12. Lipofectamine 2000 was also reported in U2OS cells. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Add DNA-lipid complexes to cells. Regarding DNA transfection, plasmids were transiently transfected with polyethylenimine (PEI) (Polysciences, Warrington, PA, USA) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). 25% cell viability respectively. Each reagent was used to transfect HEK 293, HeLa, LNCaP, HepG2, and A549 cell lines in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. We describe this new technology and how this simple versatile reagent delivers efficient, reproducible transfection of both DNA and RNA into a wide range of hard-to-transfect, biologically relevant cell models. Fair warning that sometimes the recommended protocols from Life Tech aren't that great. Specifically, when utilizing GP3K as the transfection delivery molecule, expression levels of a b- galactosidase encoding plasmid are 20-fold higher than those achieved using Lipofectamine® 2000 (LF2K) (Invitrogen Corporation). , high-speed sorting), and cells with. Therefore: 1. Increase the voltage to 100–150 V to finish the run in about 1 hr. Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. I am using the standard protocol for lipofectamine RNAiMAX (Plate cells to 60-80% confluency, Dilute 9µL lipofectamine in 150µL Opti-MEM, Dilute 30 pmol siRNA in 150µL Opti-MEM. We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. ルシフェラーゼの発現および細胞生存性はトランスフェクション24時間後(Lipofectamine ® 2000の場合)または24時間後および48時間後(Lipofectamine ® 3000の場合)にONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assayを用いて測定した。. Reagents: 10% FBS in OptiMEM, 1x PBS, OptiMEM, Lipofectamine 2000 Protocol: 1. 3000 (GP3K), we demonstrate a significant increase in transfection of RAW 264. 000 cells/well) 90% confluency at the day of transfection (100% at the day of analysis) 53 ng and 82 ng of MC are equimolar to 82 ng and 126 ng of pCMV, respectively. jetPRIME ® is a powerful in vitro transfection reagent that requires a small amount of reagent and plasmid DNA, making its use very cost-effective (Table 1). 试剂实验方案 Lipofectamine 3000出版物编号:100022234 版本号:MAN0009872 Rev B. Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid DNA is a hydrophilic molecule with a highly negative surface charge due to the presence of phosphate groups on. Mouse antiDJ1FORM This monoclonal antibody is supplied as a 1 mL aliquot of tissue culture supernatant containing 0. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. 3000 (GP3K), we demonstrate a significant increase in transfection of RAW 264. The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA, transfection reagent/method, target cell line, and number of cells Genetic Material DNA Quality and Quantity Factors Affecting Transfection 8. Plate cells so they will be 70–90% confluent at the time of transfection. Lipofectamine 3000 reagent is suitable for novel genome editing applications. ‘Bright Yellow-2’ (BY2) protoplasts. It increases the likelihood of successful cleavage and recombination with TALENs or CRISPRs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Transfection by Lipofectamine (using Invitrogen LipofectamineTM) 1. 7 cm) format SDS-PAGE gel, along with molecular weight markers. Lipofectamine® 3000试剂的脂质体用量在很宽的动态范围内均能保持很高的转染效率,可按需进行简便快速的条件优化。在需要将细胞损伤降至*的相关应用中,建议使用低毒的脂质体用量(24孔板每孔使用0. A549 (A) or MDCK (B) cells were transfected with luciferase encoding plasmid DNA using either TransIT-X2® (Mirus Bio), Lipofectamine® 2000 (Thermo Fisher Scientific) or Lipofectamine® 3000 (Thermo Fisher Scientific) for 24 hours at indicated reagent-to-DNA ratios or reagent-to-P3000™-to-DNA ratio. 当前位置: 文档下载 > 所有分类 > 自然科学 > 生物学 > Life Technologies Lipofectamine 3000 中文说明 免费下载此文档 侵权投诉 Life Technologies Lipofectamine 3000 中文说明. Lipofectamine 2000. OptiMEM(in uL, equal volume for DNA and Lipofectamine) = Lipofectamine(in uL) * 25; Protocol (6 well dish) Plate cells and grow to ~70% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG; use 1 mL for 12 well plate, and use half the amount of DNA) For each well prepare one tube for DNA and one tube for Lipofectamine:. 7ug of vsvg, and 47. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hard-to-transfect and common cells (e. Lipofectamine 2000 may be mixtures of lipofectin and lipofectamine contain DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,dimethyl -1 propanaminium trifluoroacetate), DOTMA and DOPE. The superior transfection performance allows you to reduce the reagent dose and improve cell viability. 求lipofectamine 2000中文说明书 ----英文的我有了,看不动! 回答:" T:" 不同的细胞已及转不同面积的孔板操作起来略有不同,以转染六孔板的贴壁. transfection reagent Lipofectamine® 3000. Regarding DNA transfection, plasmids were transiently transfected with polyethylenimine (PEI) (Polysciences, Warrington, PA, USA) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Prepare plasmid DNA-lipid complexes. 5 % FBS to the transfection complexes, once DNA and lipofectamine is mixed and incubated. 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Kaludercic, University of Padova (Italy) "I was very happy with the results. Lipofectamine™ 2000 Reagent should not be frozen. 24 h after transfection, cells. It belongs to geneart crispr products and services category. Lipofectamine 3000 or Lipofectamine 2000 treatment also activated critical regulators such as STAT1 and STAT3 in the type I IFN signaling pathway (Figure 1c). Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. For RNaseH1 transfections, 3 × 106 TC32 cells were transfected with 13 μg of plasmid DNA using Amaxa nucleofection (program X-001). Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Cationic liposome-mediated transfection (lipofection) has been used as an efficient method for direct gene transfer both in vitro and in vivo. We describe this new technology and how this simple versatile reagent delivers efficient, reporducible transfection of both DNA and RNA ino a wide range of hard-to-transfect, biologically relevant cell models. Thermo Fisher Scientific offers wide range of products under invitrogen brand which includes lipofectamine 3000 transfection reagent. I intend to use pX330 to express the sgRNA and the Cas9, so they are already as equimolar as I can make them. 求lipofectamine 2000中文说明书 ----英文的我有了,看不动! 回答:" T:" 不同的细胞已及转不同面积的孔板操作起来略有不同,以转染六孔板的贴壁. The topology (linear or supercoiled) and the size of the plasmid DNA vector influence the transfection efficiency. Transfection was performed in BJ fibroblasts using the Invitrogen ™ Neon Transfection System at the recommended conditions and Lipofectamine 3000 reagent at 3. A low toxicity lipid dose (0. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. Researchers use Lipofectamine™ 2000 Reagent for siRNA- and shRNA-based gene knockdown experiments, as well as for gene expression studies. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of material. This reagent delivers superior transfection efficiency and improved cell viability for the wide. 5 μl P3000 were diluted in 100 μl Optimem. A western blot was performed to determine the level of expression using -actin as a control. The difference between DOTAP and Lipofectamine 2000 may reside on the different head groups that in the latter case, consist of a polyamine moiety. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. For your convenience, the essential components of this protocol are now available in the Gibco™ Liver Cancer Starter Kit. Human iPSC-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived Mesencymal Stem Cells and Huntington's Disease (HD)-specific iPSCs plated in 24-well plates were transfected with DNA-In® Stem, Lipofectamine®2000 (L2000) and 3000 (L3000) using various amounts (µl) of reagent. I am using the standard protocol for lipofectamine RNAiMAX (Plate cells to 60-80% confluency, Dilute 9µL lipofectamine in 150µL Opti-MEM, Dilute 30 pmol siRNA in 150µL Opti-MEM. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. Lipofectamine 3000 Transfection Kit, supplied by Thermo Fisher, used in various techniques. The word transfection is a portmanteau of trans-and infection. 5 μl lipofectamine 3000 was diluted in 100 μl Optimem. Have you optimized the transfection condition with regard to 1) DNA/lipid ratios, 2) cell density, and 3) DNA dosages? Other options will be LipofectAMINE 2000 (Invitrogen), transfectam (Promega). Transfection of cells with plasmid DNA is well optimized, and the procedure is simple and quick. The recommended density should be 95-99% at the time of transfection. Superior siRNA/miRNA delivery and gene knockdown. • Nucleic acid-Lipofectamine® 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum/antibiotic. Lipofectamine® 2000 DNA Transfection Reagent Protocol See page 2 to view a typical DNA transfection procedure. Successfully transfected cell types include:. 7ug of vsvg, and 47. For RNaseH1 transfections, 3 × 106 TC32 cells were transfected with 13 μg of plasmid DNA using Amaxa nucleofection (program X-001). Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells. Use the indicated volume of DNA and P3000 ™ Reagent with each of the two volumes of Lipofectamine ® 3000 (when. Transient transfection is most efficient with supercoiled plasmid DNA. com Staff Single-Sign-On. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. A few applications that popularly utilize co-transfection are virus production, protein-protein interaction studies, stable cell line generation, or simple addition of reporter DNA constructs to normalize experimental output. Bioz Stars score: 99/100, based on 2 PubMed citations. Lipofectamine 3000 reagent for iPSC reprogramming. 6 μL per well. TransIT-X2® Dynamic Delivery System Outperforms Lipofectamine® Reagents. Lan Zhou 11 /01/00, Edited by TCH 1/21/02. 02% transfection efficacy, similar to the group of Lipofectamine 3000 & P3000 treatment, the treatment of Lipofectamine 3000 alone recorded low transfection efficacy in most cases, with minor transfection efficacy recorded in HL60 (2. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. If you can afford it ask for a free sample of the lipofectamine 3000. Tel: 010-56371250. Lipofectamine ™ 2000 는 DNA 와 RNA 모두를 진핵 세포로 전달할 때 사용하는 제품입니다. For each hard-to-transfect cell line, we tested the delivery of Cas9 RNPs using the Neon 24-well optimization protocol (Supplementary Table 4). Successfully transfected cell types include:. 1 After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. There may be quite a few cell specific issues when it comes to this topic, in particular if your cells are adherent or suspension. Conditions were used according to the manufacturer’s recommendation for lipofectamine ® 3000 and for jetPRIME ®. There's a difference between the two protocols - lipofectamine 3000 has an extra reagent that needs to be added. lipofectamine 2000 vs 3000. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. If you continue to use this site we will assume that you are happy with it. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectamine 3000采用最先进的脂质体纳米微粒技术,提供了出众的转染性能和可重复的结果。它适用于各种难转染的细胞和常见的细胞,在保证转染效率的同时也提高了细胞存活率。. complexation with Viromer® RED, Viromer® YELLOW, Lipofectamine® 3000 and Mirus TransIT®-X2 Cells cultured in 96-well plates (10% CO 2, DMEM 10% FBS, 25. Different cell lines were transfected with a GFP reporter vector using the indicated transfection reagents. Add DNA-lipid complexes to cells. I intend to use pX330 to express the sgRNA and the Cas9, so they are already as equimolar as I can make them. Each tube gets 8mls of OPTI-MEM, 32ug of gag pol, 12. Lipofectamine 3000 and sirna transfection keyword after analyzing the system lists the list of keywords related and the list of websites with related content, in addition you can see which keywords most interested customers on the this website. LipofectamineŽ 2000 Cat. Add DNA-lipid complexes to cells. 7 cells, growing in RAWGM1, prior to transfection. I am using the standard protocol for lipofectamine RNAiMAX (Plate cells to 60-80% confluency, Dilute 9µL lipofectamine in 150µL Opti-MEM, Dilute 30 pmol siRNA in 150µL Opti-MEM. The topology (linear or supercoiled) and the size of the plasmid DNA vector influence the transfection efficiency. Lipofectamine 2000 may be mixtures of lipofectin and lipofectamine contain DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,dimethyl -1 propanaminium trifluoroacetate), DOTMA and DOPE. Transfection of 293 cells in 10 cm dishes *****Abbreviated protocol***** Surface area of 15 cm dish is 18 x greater than that of a 6-well dish. A western blot was performed to determine the level of expression using -actin as a control. Kaludercic, University of Padova (Italy) "I was very happy with the results. The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. This is the first time I will have used Lipofectamine 3000, previously I only used Lipofectamine 2000, and I am wondering if anyone has found an optimal Lipo 3000 / DNA ratio for this cell line. Regarding DNA transfection, plasmids were transiently transfected with polyethylenimine (PEI) (Polysciences, Warrington, PA, USA) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). There may be quite a few cell specific issues when it comes to this topic, in particular if your cells are adherent or suspension. 7 Cells with Lipofectamine 2000 AfCS Procedure Protocol PP00000182 Page 2 Preparation of RAW 264. Transfections were performed either in serum (+FBS) or without serum (−FBS). 很多文献都是用Lipofectamine 2000,但是都说2000毒性很大,转染效率不高。现在有Lipofectamine 3000,因为新产品,现在做的人少,不知道效果怎么样,所以请问用过3000的大神,哪种效果好呢?谢谢了?. Each reagent was used to transfect HEK 293, HeLa, LNCaP, HepG2, and A549 cell lines in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. Transient transfection is most efficient with supercoiled plasmid DNA. Lipofectamine® 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. TRANSIENT TRANSFECTION OF MAMMALIAN CELLS WITH LipofectAMINE. Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Make sufficient 10% FBS in Opti-MEM media (w/o antibiotics) sterile filter 2. Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. Lipofectamine 3000 and P3000™ Enhancer Reagent (Thermo Fisher Scientific Inc) were used for transient plasmid transfections according to manufacturer’s protocol and the cells were cultured for 24 h. ルシフェラーゼの発現および細胞生存性はトランスフェクション24時間後(Lipofectamine ® 2000の場合)または24時間後および48時間後(Lipofectamine ® 3000の場合)にONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assayを用いて測定した。. Lipofectamine® 3000试剂的脂质体用量在很宽的动态范围内均能保持很高的转染效率,可按需进行简便快速的条件优化。在需要将细胞损伤降至*的相关应用中,建议使用低毒的脂质体用量(24孔板每孔使用0. Lipofectamine® 2000 Reagent Protocol Outline A. Here are some suggestions for 96-well plate format: •The day before transfection, seed 25,000 MCF-7 cells per well of a 96-well plate. Wesseling´s) Notes: Volumes are per well (12-well plates) In my hands, it works up better up to DIV10-12 cultures (at the day of transfection). For each hard-to-transfect cell line, we tested the delivery of Cas9 RNPs using the Neon 24-well optimization protocol (Supplementary Table 4). In combination with Plus Reagent, it gives pretty nice results. I attached both lipofectamine and plus reagent sheets. Cells should be plated on collagen-coated dishes, as for normal propagation. This is going to be very helpful, since I have see a lot of posts describing nucleofection, but not much dealing with lipofectamine. Lipofectamine 3000 reagent is suitable for novel genome editing applications. Lipofectamine 2000 Lipofectamine 3000 Competitor 1 Competitor 2 Western blot, HepG2 GST-STAT ϐ-actin Figure 1. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Before seeding cells for virus package, make sure HEK293T cells are in rapid replication state by growth cells to approximately 75%~80% confluence in a 10 cm cell culture dish and passaging at 1:2 ratio for at least 2 consecutive days; 2. Transfecting and Plating RAW 264. 5 % FBS to the transfection complexes, once DNA and lipofectamine is mixed and incubated. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 为此,在Lipofectamine 3000的开发过程中,科学家们优化了转染过程的四个步骤,并结合了最先进的脂质体纳米微粒技术。因此,Lipofectamine 3000出色的转染性能可以让您减少试剂的用量,并降低潜在的毒性风险。 3000与2000相比,转染效率如何?. Lipofectamine® 2000 DNA Transfection Reagent Protocol See page 2 to view a typical DNA transfection procedure. General Note: Plate cells for 2-12hrs (but not over 24 hours) prior to transfection to achieve a 40-80% confluence at the time of transfection. Each reaction mix volume is for one well and accounts for pipetting variations. SiRNA silencing was performed using 100 nM siRNA and Lipofectamine® RNAiMAX Reagent (Thermo Fisher Scientific Inc) according to manufacturer. Campaign goals The goal of this campaign was to drive trial and adoption of Lipofectamine® 3000, a versatile transfection reagent with up to 10-fold higher transfection efficiency in difficult-to-transfect cell types. HIV CMVeGFP Virus was produced in HEK 293FT cells using either CaPO 4, Lipofectamine® 2000 or TransIT®-Lenti Transfection Reagent per the manufacturer's protocol. This is the first time I will have used Lipofectamine 3000, previously I only used Lipofectamine 2000, and I am wondering if anyone has found an optimal Lipo 3000 / DNA ratio for this cell line. Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. Use the indicated volume of DNA and P3000 ™ Reagent with each of the two volumes of Lipofectamine ® 3000 (when. ,unpublished observations) have revealed that, the detection time may be too brief (e. To identify the best transfection reagents for delivery of Cas9 RNPs, initially we compared several commercially available protein transfection reagents, including Lipofectamine 2000, Lipofectamine 3000, Lipofectamine RNAiMAX, Lipofectamine MeassengerMax, TurboFect, and Xfect Protein transfection reagent. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX Overview: Cas9 nuclease may be used in vivo to create targeted genome modifications. In this study, we demonstrated that using an improved transfection reagent, such as Lipofectamine® 3000, helps to eliminated many of the pain points. I intend to use pX330 to express the sgRNA and the Cas9, so they are already as equimolar as I can make them. Lipofectamine 3000 can be used to transfect a broad spectrum of cell line with nucleic acids. A549 (A) or MDCK (B) cells were transfected with luciferase encoding plasmid DNA using either TransIT-X2® (Mirus Bio), Lipofectamine® 2000 (Thermo Fisher Scientific) or Lipofectamine® 3000 (Thermo Fisher Scientific) for 24 hours at indicated reagent-to-DNA ratios or reagent-to-P3000™-to-DNA ratio. 11668-019 Size: 1. 11668-027 Size: 0. There's a difference between the two protocols - lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. Euplotes transfection using Lipofectamine 3000 (provisional) protocol (method) by Angela Piersanti. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hard-to-transfect and common cells (e. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. The difference between DOTAP and Lipofectamine 2000 may reside on the different head groups that in the latter case, consist of a polyamine moiety. Chemical Supplier Product List. ZERO BIAS - scores, article reviews, protocol conditions and more. In the present study, we demonstrated that Lipofectamine® 3000 can be used to. TransIT-X2® Dynamic Delivery System Outperforms Lipofectamine® Reagents. TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent MCF10A Breast cancer cells Complete growth medium Component Cat. 7ug of vsvg, and 47. Component 96-well 24-well 6-well. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 为此,在Lipofectamine 3000的开发过程中,科学家们优化了转染过程的四个步骤,并结合了最先进的脂质体纳米微粒技术。因此,Lipofectamine 3000出色的转染性能可以让您减少试剂的用量,并降低潜在的毒性风险。 3000与2000相比,转染效率如何?. Use the indicated volume of DNA and P3000 Reagent with each of the two volumes of Lipofectamine 3000 (when performing optimization). Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation. Mix well by pipetting. Lipofectamine® 3000 Transfection Reagent Protocol Transfect cells according to the following table. Lipofectamine® 2000 DNA Transfection Reagent Protocol See page 2 to view a typical DNA transfection procedure. The recommended density should be 95-99% at the time of transfection. TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent LNCaP Prostate cancer cells Complete growth medium Component Cat. Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Lipofectamine 2000 Transfection for HEK293 cells (about 60-70% transfection rate) Transfect cells (6-well plate): 1) HEK 293 cells should be 85-95% confluent (seed 600,000 cells into each well of a 6-well plate on Day 1, transfect on Day 2, then allow to grow on Day 3, harvest on Day 4). Increase the voltage to 100–150 V to finish the run in about 1 hr. Use 45 ug of plasmid DNA. Include a control. For each hard-to-transfect cell line, we tested the delivery of Cas9 RNPs using the Neon 24-well optimization protocol (Supplementary Table 4). Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. 8/1/2017 Lipofectamine 3000 Ab G Lipofectamine 3000 can be used to transfect a broad spectrum of cell line with nucleic acids. complexation with Viromer® RED, Viromer® YELLOW, Lipofectamine® 3000 and Mirus TransIT®-X2 Cells cultured in 96-well plates (10% CO 2, DMEM 10% FBS, 25. For plasmid transfection the best method that can be used is lipofectamine. Let sit at RT for 5 minutes. We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. Each reaction mix volume is for one well and accounts for pipetting variations. This is going to be very helpful, since I have see a lot of posts describing nucleofection, but not much dealing with lipofectamine. Component 96-well 24-well 6-well. I will probably still need to optimize the protocol, but I got quite good results with Viromer. The difference between DOTAP and Lipofectamine 2000 may reside on the different head groups that in the latter case, consist of a polyamine moiety. Dilute 3 µg DNAs and 1 µg reporter DNAs (fluorescence proteins) into OptiMEM or DMEM without FBS and mix gently. Each cell line was transfected with a luciferase reporter plasmid using either ViaFect™ Transfection Reagent, Lipofectamine® 2000 Transfection Reagent or Lipofectamine® 3000 Transfection Reagent following manufacturer instructions, applying either 5µl (50ng of DNA) or 10µl (100ng of DNA) of the transfection complex in each well of a 96. lifetechnologies. The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA, transfection reagent/method, target cell line, and number of cells Genetic Material DNA Quality and Quantity Factors Affecting Transfection 8. The word transfection is a portmanteau of trans-and infection. The following day, cells were transfected with 2 µg of SETD2 plasmid or pcDNA3. Enhanced protein expression using Lipofectamine. We describe this new technology and how this simple versatile reagent delivers efficient, reproducible transfection of both DNA and RNA into a wide range of hard-to-transfect, biologically relevant cell models. Lipofectamine® 3000 Reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells Important Guidelines ∤ Make DNA-Lipofectamine® 3000 complexes in serum-free medium such as Opti-MEM® Reduced Serum Medium and add directly to cells in culture. vs clipart. 1 After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. Lipofectamine 3000 采用*进的脂质体纳米微粒技术,提供了出众的转染性能和可重复的结果。它适用于各种难转染的细胞和常见的细胞,在保证转染效率的同时也提高了细胞存活率。. General Note: Plate cells for 2-12hrs (but not over 24 hours) prior to transfection to achieve a 40-80% confluence at the time of transfection. The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Add the Lipofectamine reagent dropwise and mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing). Discover the versatile reagent with the power to transfect your most difficult cells for just pennies per reaction The Invitrogen™ Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. I, personally, have used this reagent to transfect cells for fluorescent microscopy, GST pull downs, and co-immunoprecipitation experiments. lipofectamine 3000 Lipofectamine 3000 Reagent | Thermo … Lipofectamine® 3000 reagent was developed for high-efficiency transfection and improved cell viability in the broadest range of cell types. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. Store at 2–8°C. Identification of Lipofectamine CRISPRMAX. Yes, you can avoid the poly-d-lysine coating step when using Lipofectamine 3000 as your transfection reagent to produce LV lentivirus. I am looking forward to the suggestions for increasing the transfection efficiency of IMR-32 to a higher. This reagent delivers superior transfection efficiency and improved cell viability for the wide. Lipofectamine® 3000 Transfection Reagent Protocol Transfect cells according to the following table. transfection reagent Lipofectamine® 3000. Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Identification of Lipofectamine CRISPRMAX. Human iPSC-derived neural stem cells (NSC), Human embryonic stem cells, adipose-derived Mesencymal Stem Cells and Huntington's Disease (HD)-specific iPSCs plated in 24-well plates were transfected with DNA-In® Stem, Lipofectamine®2000 (L2000) and 3000 (L3000) using various amounts (µl) of reagent. 7 Cells for Transfection The following is a procedure for harvesting RAW 264. Gentle with low toxicity Lipofectamine® 3000 reagent was designed to optimize every step in the transfection process. Studies were undertaken to evaluate and compare the transfection efficacy. If there appears to be large-scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates. 5 ml Contents and Storage LipofectamineŽ 2000 is supplied in liquid form at a concentration of 1 mg/ml. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Add the 3 plasmid mix dropwise to the diluted Lipofectamine reagent and mix by swirling the tip or gently flicking the tube. In addition to being a top performing reagent for difficult cell types, Invitrogen™ Lipofectamine™ 3000 Transfection Reagent is a highly efficient, cost-effective tool for lentiviral production. Lipofectamine 2000 Transfection for HEK293 cells (about 60-70% transfection rate) Transfect cells (6-well plate): 1) HEK 293 cells should be 85-95% confluent (seed 600,000 cells into each well of a 6-well plate on Day 1, transfect on Day 2, then allow to grow on Day 3, harvest on Day 4). Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). ,unpublished observations) have revealed that, the detection time may be too brief (e. Lipofectamine 3000 Transfection Kit, supplied by Thermo Fisher, used in various techniques. Especially Lipofectamine 3000, as it is a most efficient versatile reagent for the widest range of cell types including d….