Lipofectamine 3000


Add ~2g of DNA to Eppendorf tube in the hood. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Lipofectamine® 3000,C ntains one vial of Lipofectamine® 3000 Reagent and one vial of P3000TM Enhancer Reagent. Conditions were used according to the manufacturer's recommendation for lipofectamine ® 3000 and for jetPRIME ®. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array 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. 2016-03-21 lipofectamine 3000 转染培养基中是否可以含 2016-07-03 龙泉刀剑的章氏和查氏哪家日本刀做的好啊 2000-3000价 2017-11-22 rnaimax和lipofectamine 2000的区别; 2013-04-04 给国外汇款选择哪个银行手续费最低?电汇3000美金. Store at +4°C. TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent LNCaP Prostate cancer cells Complete growth medium Component Cat. HepG2 cells were transfected with a vector expressing GST-tagged STAT protein. If you can afford it ask for a free sample of the lipofectamine 3000. The transfection with 1, 2, and 4 μl Lipofectamine 2000 resulted in approximately 87% eGFP positive cells. I will probably still need to optimize the protocol, but I got quite good results with Viromer. 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. In our lab, we use Lipofectmamine 3000 reagent as our primary transfection reagent for a wide range of experiments. 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. RNAi, Oligos, Assays, Gene Editing & Gene Synthesis Tools. Package of Lenti-shRNA virus using HEK293T cells 1. However, the activation of extracellular signal-regulated kinase and IjBa was not increased by Lipofectamine 3000 or Lipofectamine 2000 treatment (Figure 1d). For RNaseH1 transfections, 3 × 106 TC32 cells were transfected with 13 μg of plasmid DNA using Amaxa nucleofection (program X-001). 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. 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. Transfection of PC12 Cells with Lipofectamine. Successfully transfected cell types include:. 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 is a transfection reagent that was developed to improve transfection efficiency in many difficult to transfect cell types. Transfection using Lipofectamine 3000 was performed according to the manufacturer's protocol with a DNA to Lipofectamine ratio of 1 : 3 w/v. 1, respectively, with lipofectamine 3000 by following the manufacturer's protocol. For your convenience, the essential components of this protocol are now available in the Gibco™ Liver Cancer Starter Kit. All transfections were carried out using Lipofectamine RNAiMax or Lipofectamine 3000 (Invitrogen) following the manufacturer's instructions. Transfection of NIH3T3 cells, Hela, Swis 3T3, 293T with Lipofectamine 2000 1. i am going to transfect my 293 cells for the production of Adenovirus so i would like to know your opinions about it. Yes, you can avoid the poly-d-lysine coating step when using Lipofectamine 3000 as your transfection reagent to produce LV lentivirus. Lipofectamine 2000 Transfection Reagent is best transfection reagent available from Life Technologies for the co-transfection of siRNA and plasmid DNA. SiRNA silencing was performed using 100 nM siRNA and Lipofectamine® RNAiMAX Reagent (Thermo Fisher Scientific Inc) according to manufacturer. Mix and let stand 20 minutes prior to addition to. We have had success using Lipofectamine reagents in the past and the Lipofectamine 3000 appears to be the best option. 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:. Different cell lines were transfected with a GFP reporter vector using the indicated transfection reagents. 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?. I did get the knockdown to work when using my siRNA with lipofectamine 2000, however. 7 cm) format SDS-PAGE gel, along with molecular weight markers. General Protocol for Western Blotting Protein separation by gel electrophoresis 1. Studies were undertaken to evaluate and compare the transfection efficacy. Lipofectamine 2000. IMLF can be transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) but transfection efficiency is higher using recombinant adenovirus (described later), which gives very good expression in a short-time relative to other methods. miRNA-30a functions as a tumor suppressor by downregulating cyclin E2 expression in castration-resistant prostate cancer. 5 ml Contents and Storage LipofectamineŽ 2000 is supplied in liquid form at a concentration of 1 mg/ml. Plate cells so they will be 70–90% confluent at the time of transfection. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 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. Make sufficient 10% FBS in Opti-MEM media (w/o antibiotics) sterile filter 2. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. Gibco™ Ham’s F-12 Nutrient Mix 11765054 5% Horse Serum –. Cost-effective: less DNA and less reagent. Lipofectamineなどの遺伝子導入試薬が、遺伝子導入効率を高めるメカニズムを教えてください。Lipofectamine 2000などの遺伝子導入試薬が、transfection efficiencyなどを高める試薬であることはわかっていますが、マニュアルを見てもなぜそうなるのかと云うメカニズムの記述が見つかりませんでした. Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). Use 144 ul of Lipofectamine 2000. However, you still need to do an optimization on these reagents. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. 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 Make DNA-Lipofectamine® 3000 complexes in serum-free medium such as Opti-MEM® Reduced Serum Medium and add directly to cells in culture medium, in the presence or. Fair warning that sometimes the recommended protocols from Life Tech aren't that great. Lipofectamine 3000 Reagent Protocol Lipofectamine 3000 Transfection Reagent Protocol Transfect cells according to the following table. 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. Standard versus Reverse Transfection. 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). The topology (linear or supercoiled) and the size of the plasmid DNA vector influence the transfection efficiency. Optimized DNA delivery in Stem Cells. Transfecting siRNA using Lipofectamine™ RAiAX Pin Liu, 2017 Declaration: The protocal is from Thermo Fisher Scientific Important Guidelines for Transfection 1. " Review date: 03 Feb 2017 | Invitrogen™ Lipofectamine® 3000 Transfection Reagent. 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?. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Add the Lipofectamine reagent dropwise and mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing). Lipofectamine 3000 and RNAiMAX outperformed Lipofectamine 2000 in HEK 293 cells (Figure S1), which is in agreement with the recent finding that RNAiMAX performed better than Lipofectamine 2000 for delivery of Cas9 RNPs with low cell toxicity (Zuris et al. - Characterization of transfection efficiency of IMR32 through calcium phosphate and lipofectamine 3000 transfection. Tel: 010-56371250. 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. 3000 (GP3K), we demonstrate a significant increase in transfection of RAW 264. 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 Make DNA-Lipofectamine® 3000 complexes in serum-free medium such as Opti-MEM® Reduced Serum Medium and add directly to cells in culture medium, in the presence or. Enhanced protein expression using Lipofectamine ® 3000 reagent. Lipofectamine® 2000 Reagent Protocol Outline A. P 7170 For Cellulose Acetate Protein Detection and Western Blot Total Protein Detection. vs clipart. Lipofectamine ™ Reagents Go online to view related products. 7 cm) format SDS-PAGE gel, along with molecular weight markers. Use the indicated volume of DNA and P3000 ™ Reagent with each of the two volumes of Lipofectamine ® 3000 (when. Lipofectamine 3000 と一緒にはいっているP3000 reagentはどんな組成、役割がありますか? ご存知の方がいらっしゃれば、お答えいただけると幸いです。. 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. I will probably still need to optimize the protocol, but I got quite good results with Viromer. I attached both lipofectamine and plus reagent sheets. 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 Make DNA-Lipofectamine® 3000 complexes in serum-free medium such as Opti-MEM® Reduced Serum Medium and add directly to cells in culture medium, in the presence or. "Dilute Lipofectamine™3000 Reagent in Opti-MEM™ Medium (2 tubes), mix well" Follow the instructions as indicated. TRANSIENT TRANSFECTION OF MAMMALIAN CELLS WITH LipofectAMINE. Transfection of cells with plasmid DNA is well optimized, and the procedure is simple and quick. com/3000 A persistent challenge in genome editing is really obtaining a high transfection efficiency that is reprod. View our Transfection products at Fisher Scientific. Guaranteed stable for 12 months when properly stored. Transfection was performed in BJ fibroblasts using the Invitrogen ™ Neon Transfection System at the recommended conditions and Lipofectamine 3000 reagent at 3. , HEK293, HeLa). Enhanced protein expression using Lipofectamine ® 3000 reagent. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells. Euplotes transfection using Lipofectamine 3000 (provisional) protocol (method) by Angela Piersanti. lipofectamine holds a protocol for transfecting suspenson cell line. The most common sizes factor are listed below. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hard-to-transfect and common cells (e. 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). This reagent delivers superior transfection efficiency and improved cell viability for the wide. HepG2 cells were transfected with a vector expressing GST-tagged STAT protein. Invitrogen™ Lipofectamine™ 3000 Transfection Reagent can help improve lentivirus production. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. View our Transfection products at Fisher Scientific. Each tube gets 8mls of OPTI-MEM, 32ug of gag pol, 12. Use 45 ug of plasmid DNA. Using 4 μl Lipofectamine 2000, a Geo Mean of 210 was detected. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells. 7 cells, growing in RAWGM1, prior to transfection. TRANSIENT TRANSFECTION OF MAMMALIAN CELLS WITH LipofectAMINE. Lipofectamine® 3000 is a transfection reagent that was developed to improve transfection efficiency in many difficult to transfect cell types. Guaranteed stable for 12 months when properly stored. 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. Mix each tube well by vortexing or flicking the tube. 7ug of appropriate viral plasmid. Lipofectamine® 2000 Reagent Protocol Outline A. Lan Zhou 11 /01/00, Edited by TCH 1/21/02. The following day, cells were transfected with 2 µg of SETD2 plasmid or pcDNA3. The normalized GFP intensity was used as an indicator of transfection efficiency. transfection reagent Lipofectamine® 3000. 7ug of appropriate viral plasmid. Lipofectamine ™ 2000 는 DNA 와 RNA 모두를 진핵 세포로 전달할 때 사용하는 제품입니다. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. Scaling Up or Down Lipofectamine® 3000 Reagent Transfections Use the following table to scale the volumes for your transfection experiment. We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. 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. Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. 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. Standard versus Reverse Transfection. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. 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. This is going to be very helpful, since I have see a lot of posts describing nucleofection, but not much dealing with lipofectamine. Euplotes transfection using Lipofectamine 3000 (provisional) protocol (method) by Angela Piersanti. Lipofectamine™ 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Terms of Service; Privacy Policy; Testimonial; FAQ; Pricing. Thermo Fisher Scientific is the world leader in serving science, with revenues of more than $24 billion and approximately 70,000 employees globally. Lipofectamine™ 2000 Reagent should not be frozen. com Staff Single-Sign-On. Add 1µg of BAC DNA to each tube and mix by flicking tube 4. Prepare plasmid DNA-lipid complexes. 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. The complex was added to HEK293 cells (around 70-80% confluent) in 35-mm dish. Each reagent was used to transfect HEK 293, HeLa, LNCaP, A549, and HepG2 cells in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. 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などの遺伝子導入試薬が、遺伝子導入効率を高めるメカニズムを教えてください。Lipofectamine 2000などの遺伝子導入試薬が、transfection efficiencyなどを高める試薬であることはわかっていますが、マニュアルを見てもなぜそうなるのかと云うメカニズムの記述が見つかりませんでした. Its systems, consumables and services enable researchers to accelerate scientific exploration. Transfection is the process by which nucleic acids are introduced into mammalian cells. 7 cm) or midi (13. Comparison of CaPO 4, Lipofectamine® 2000 or TransIT®-Lenti Generated Lentivirus. Add DNA-lipid complexes to cells. 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?. Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. Scaling Up or Down Lipofectamine® 3000 Reagent Transfections Use the following table to scale the volumes for your transfection experiment. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells. Plate cells so they will be 70–90% confluent at the time of transfection. , HEK293, HeLa). Transfection of cells with plasmid DNA is well optimized, and the procedure is simple and quick. (PEI) or Lipofectamine 2000 (Invitrogen) transfection 2. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Company Name: Beijing Solarbio Science & Tecnology Co. We also found that hematopoietic cells were hard to transfect using Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine CRISPRMAX. In this study, we demonstrated that using an improved transfection reagent, such as Lipofectamine® 3000, helps to eliminated many of the pain points. The normalized GFP intensity was used as an indicator of transfection efficiency. Successfully transfected cell types include:. Add the 3 plasmid mix dropwise to the diluted Lipofectamine reagent and mix by swirling the tip or gently flicking the tube. Each reagent was used to transfect HEK 293, HeLa, LNCaP, A549, and HepG2 cells in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. 3-fold in. SCR510 ; The STEMCCA Constitutive Polycistronic Lentivirus Kit contains high titer polycistronic lentivirus & Polybrene transfection reagent that have been validated for the generation of mouse & human induced pluripotent stem cells. 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). TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent LNCaP Prostate cancer cells Complete growth medium Component Cat. 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. Late embryonic primary rat astrocytes and adult rat Schwann cells were transfected 24 hr after seeding in the presence of serum with a pMaxGFP plasmid using either Lipofectamine 3000 or Viromer RED. 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. 7 Manual siRNA Transfection for Infection assay OR lysate collection. Our prior studies (Stewart et al. Standard versus Reverse Transfection. 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. Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. 2) In a separate well, mix 25ul OptiMEM with 1ul Lipofectamine; incubate 15 min at RT. Lipofectamine 3000-Reagenz eignet sich für Novel Genome Editing-Anwendungen. Add the Lipofectamine reagent dropwise and mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing). 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. 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. 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。. Depending on the cell line, there may be some cytotoxicity associated with the reagents. In general, except in MCF-7 cells, which showed a 45. 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. RNAi, Oligos, Assays, Gene Editing & Gene Synthesis Tools. There are several ways in which to introduce Cas9-guide RNA complexes into cells. Transfection of PC12 Cells with Lipofectamine. Dilute 3 µg DNAs and 1 µg reporter DNAs (fluorescence proteins) into OptiMEM or DMEM without FBS and mix gently. 25% cell viability respectively. For the cell lines easy to transfection, you can use either of the procedures (Reverse or Forward. 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). 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). Bioz Stars score: 99/100, based on 2 PubMed citations. From the luciferase activities and consistencies of data using different transfection reagents including GeneJammer, Effectine, Wellfect, Lipofectamine, what I can say from the data I observed, the efficiency of transfection is much more better in HCT116 cells using Lipofectamine 2000. Store at +4°C. Component 96-well 24-well 6-well. 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. Our mission is to enable our customers to make the world healthier, cleaner and safer. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Lipofectamine 2000. Superior siRNA/miRNA delivery and gene knockdown. 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. ” —Julien Sebag, PhD Assistant Professor, Vanderbilt University 2 Life Technologies™ | Lipofectamine 3000 reagent ® 目次 患者ドナー由来線維芽細胞からのiPSC樹立 4 多能性幹細胞のゲノム編集 6 Lipofectamine® 3000を用いた 多能性幹細胞へのトランスフェクション 7 ヒト線維芽細胞リ. (PEI) or Lipofectamine 2000 (Invitrogen) transfection 2. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. 7 cells, growing in RAWGM1, prior to transfection. 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. Gibco™ RPMI 1640 Medium, with GlutaMAX™ Supplement 61870036 10% Gibco™ FBS A3160401 1 mM Gibco™ Sodium Pyruvate 11360070 Proper culture techniques and procedures are an essential part of ensuring successful. Different cell lines were transfected with a GFP reporter vector using the indicated transfection reagents. Use the indicated volume of DNA and P3000 ™ Reagent with each of the two volumes of Lipofectamine ® 3000 (when. ‘Bright Yellow-2’ (BY2) protoplasts. In combination with Plus Reagent, it gives pretty nice results. It belongs to geneart crispr products and services category. Add DNA-lipid complexes to cells. The word transfection is a portmanteau of trans-and infection. Add 2-3 ml PBS to wash 4. Lipofectamine 3000 Reagent Protocol Lipofectamine 3000 Transfection Reagent Protocol Transfect cells according to the following table. Successfully transfect the. I personally have used this reagent to transfect cells for fluorescent microscopy, GST pull downs, and co-immunoprecipitation experiments. 1 After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. We use cookies to ensure that we give you the best experience on our website. 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 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. This reagent delivers superior transfection efficiency and improved cell viability for the wide. 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). Lipofectamine 3000 reagent for iPSC reprogramming. Especially Lipofectamine 3000, as it is a most efficient versatile reagent for the widest range of cell types including d…. 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. Place each into 2. I was using Lipofectamine® to transfect HEK cells and was only getting about 20% transfection rates but after using TransIT®-2020 it went from 20% to 70%! I highly recommend using this reagent. semanticscholar. Lipofectamine 2000 Transfection Reagent is best transfection reagent available from Life Technologies for the co-transfection of siRNA and plasmid DNA. Each reagent was used to transfect HEK 293, HeLa, LNCaP, A549, and HepG2 cells in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. Let sit at RT for 5 minutes. 11668-027 Size: 0. I attached both lipofectamine and plus reagent sheets. Add DNA-lipid complexes to cells. 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). According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. Transfection of cells with plasmid DNA is well optimized, and the procedure is simple and quick. Therefore: 1. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells. Lipofectamine 3000 reagent is suitable for novel genome editing applications. What is the appropriate way of adding lipofectamine 3000 to cells? Is it added after removing media from each well, and then adding the lipo and DNA mixture dropwise to the well, incubating for 6. 5mL centrifuge tube for each transfection and add 50µL opti-MEM media to each. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. There's a difference between the two protocols - lipofectamine 3000 has an extra reagent that needs to be added. Unlike other commonly used transfection reagents that are used in Lentivirus production, Lipofectamine 3000 actually requires a high cell density (cell type: 293T or FT) at the time of transfection. LipofectamineŽ 2000 Cat. I am looking forward to the suggestions for increasing the transfection efficiency of IMR-32 to a higher. Protocols and techniques vary widely and include lipid transfection and chemical and physical methods such as electroporation. Lipofectamineなどの遺伝子導入試薬が、遺伝子導入効率を高めるメカニズムを教えてください。Lipofectamine 2000などの遺伝子導入試薬が、transfection efficiencyなどを高める試薬であることはわかっていますが、マニュアルを見てもなぜそうなるのかと云うメカニズムの記述が見つかりませんでした. 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). Consistent with this, the. 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. SiRNA silencing was performed using 100 nM siRNA and Lipofectamine® RNAiMAX Reagent (Thermo Fisher Scientific Inc) according to manufacturer. Lipofectamine 2000. - Stable transfection of HeLa cells with transposon containing NMYC (obtained through subcloning) as a system for studying the effect of NMYC-CRISPR on cancer cells. here is the protocol for lipofectamine and suspension cell line Transfecting Suspension Mammalian Cells. 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. From the luciferase activities and consistencies of data using different transfection reagents including GeneJammer, Effectine, Wellfect, Lipofectamine, what I can say from the data I observed, the efficiency of transfection is much more better in HCT116 cells using Lipofectamine 2000. Life Technologies is a global biotechnology tools company dedicated to improving the human condition. 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). I was recently having issues using lipofectamine 3000 so did a lot of troubleshooting. 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. Bioz Stars score: 99/100, based on 725 PubMed citations. 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. Euplotes transfection using Lipofectamine 3000 (provisional) protocol (method) by Angela Piersanti. In addition to higher transfection efficiency (% GFP+ cells) and higher expression level (brighter) as compared to those of Lipofectamine® 3000, HUVEC Avalanche® Transfection Reagent also shows the least toxicity to the cells. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Using lipofectamine 3000 reagents, I am getting only about 30% efficiency for transfection. Add 1µg of BAC DNA to each tube and mix by flicking tube 4. YELLOW, Lipofectamine® 2000 and Lipofectamine® 3000 >> around 50% efficiency with standard protocol and lowest concentration of Viromer Data from N. Include a control. Especially Lipofectamine 3000, as it is a most efficient versatile reagent for the widest range of cell types including d…. Lan Zhou 11 /01/00, Edited by TCH 1/21/02. The protocol for Lipofectamine 3000 LV production requires high cell density, 95-99% confluence at the time of transfection. 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. However, they can be difficult to produce. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Successfully transfected cell types include:. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Each reagent was used to transfect HEK 293, HeLa, LNCaP, A549, and HepG2 cells in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. Life Technologies is a global biotechnology tools company dedicated to improving the human condition. It was very easy to follow the protocol and I obtained high cell viability. For plasmid transfection the best method that can be used is lipofectamine. 75µL for 24-well plates) is suggested for applications requiring minimal disruption of the cells. Identification of Lipofectamine CRISPRMAX. 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. This video shows you how to pronounce Lipofectamine. Aspirate off PBS 5. Aspirate medium off cells 3. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of material. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Cost-effective: less DNA and less reagent. Lipofectamine 3000: 4ul. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. 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. 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). 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. Yes, you can avoid the poly-d-lysine coating step when using Lipofectamine 3000 as your transfection reagent to produce LV lentivirus. Oncology Letters; International Journal of Oncology; Molecular and Clinical Oncology; Experimental and Therapeutic Medicine; International Journal of Molecular. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. I personally have used this reagent to transfect cells for fluorescent microscopy, GST pull downs, and co-immunoprecipitation experiments. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Lipofectin and RNAiMAX showed lower cytotoxicity to Huh-7 cells, with 75. Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. 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:. Dilute 3 µg DNAs and 1 µg reporter DNAs (fluorescence proteins) into OptiMEM or DMEM without FBS and mix gently. Lipofectamine ™ 2000 한글 매뉴얼. Lipofectamine 3000 与 Lipofectamine 2000 的对比. P 7170 For Cellulose Acetate Protein Detection and Western Blot Total Protein Detection. 试剂实验方案Lipofectamine® 3000出版物编号:100022234 版本号:MAN0009872 Rev B. Transient transfection is most efficient with supercoiled plasmid DNA. Lipofectamine 2000 Lipofectamine 3000 Competitor 1 Competitor 2 Western blot, HepG2 GST-STAT ϐ-actin Figure 1. transfection reagent Lipofectamine® 3000. Cells should be plated on collagen-coated dishes, as for normal propagation. II) is specially designed and formulated by adding proprietary enhancers for transfecting HEK293 cells and other mammalian cells (Figure 1). Which is better!!! Lipofectamine or Fugene 6. This reagent delivers superior transfection efficiency and improved cell viability for the wide. 1% sodium azide. 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. Identification of Lipofectamine CRISPRMAX. Prepare plasmid DNA-lipid complexes. " Review date: 03 Feb 2017 | Invitrogen™ Lipofectamine® 3000 Transfection Reagent. ZERO BIAS - scores, article reviews, protocol conditions and more. Identification of Lipofectamine CRISPRMAX. Aspirate off PBS 5. Cationic liposome-mediated transfection (lipofection) has been used as an efficient method for direct gene transfer both in vitro and in vivo. Add 8 ml of 10% FBS/ Opti-MEM media /plate 6. 7 cells, growing in RAWGM1, prior to 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. 5 % FBS to the transfection complexes, once DNA and lipofectamine is mixed and incubated. View our Transfection products at Fisher Scientific. Successfully transfected cell types include:. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. 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 Make DNA-Lipofectamine® 3000 complexes in serum-free medium such as Opti-MEM® Reduced Serum Medium and add directly to cells in culture medium, in the presence or. Seed 500,000 pure K5 62 cells in 12 well plate, in 1mL complete RPMI media.