Genscript recommends using lipofectamine 2000 for all transfections. The mechanism of lipofectamine 2000 mediated transmembrane. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent. Mcf7 cells and adipocytes were transfected with either the sirna sequence or the negative control mimic using lipofectamine 2000. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Stealthsirna transfection protocol lipofectamine 2000. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit. In this case, the cas9 rnps and lipofectamine crisprmax reagent were prepared in two separate tubes as described above with 500 ng geneart platinum cas9 nuclease, 125 ng grna, and 1 l cas9 plus reagent in tube1, and 1. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. I use 24well plates for transfections in triplicates.
Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Although lipofectin showed the best safety profile with a cell viability of 92. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Human adipocytes stimulate invasion of breast cancer mcf7. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. The highest transfection efficiency in many cell types and formats e. A sample protocol is listed here for transfection experiments performed in 6well plates.
Invitrogen lipofectamine 3000 transfection reagent fisher. This protocol is well adjusted for cancer cell lines. Which is the best protocol to transfect mcf 7 cell line. The intracellular trafficking mechanism of lipofectamine. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Has anyone used lipofectamine 3000 for plasmid transfection. We have lipofectamine 2000, and im just going through the protocol that is suggested by said manufacturer. We found that lipofectamine 3000 has much higher dna transfection efficiency than any other transfection reagents in mcf 7 cells, the transfection efficiency reached 6080% 5kb gfp expression. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Plate cells so they will be 6080% confluent at the time of transfection.
Transfection protocol on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Altogen biosystems produces preoptimized highefficiency transfection reagent kits for the mcf 7 breast cancer cell line. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Higher densities can lead to spontaneous differentiation. Transitbrca transfection reagent is the first dedicated breast cancer cell line specific reagent available to researchers. I am looking for a protocol for plasmid transfection into mcf 10a cells. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Help on transfecting mcf7 and hct 116 cells transfection. While it delivers efficiently to many breast cancer cell types, it also maintains low cellular toxicity resulting in reduced experimental biases. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine transfection of c2c12 cells bridges lab. Add 6l of lipofectamine2000 from biostores and 100 l of. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf 7 with lipofectamine 2000 maartje vogel.
Which is the best protocol to transfect mcf7 cell line. Improved delivery of cas9 proteingrna complexes using. Hi, im going to be doing some transfections of mcf 7 and mdamb231 cells, with a plasmid containg cat, and my gene of interest. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Afcs procedure protocol pp00000182 version 1, 103103. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Cells from the mcf 7 breast cancer cell line are widely regarded as a near perfect model to examine the growth of malignant tumors. 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. As for mcf 7 cells figure 10, lipofectamine 3000 demonstrated the highest transfection efficacy 58. Lipofectamine 2000 reagent thermo fisher scientific.
Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Feb 18, 2016 a reverse transfection protocol was used to transfect mcf7 and hepg2 cells. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when. Lipofectamine 2000 transfection protocol 10cm plates reagents. At the moment we are using lipofectamine ltx plus, but the transfection efficiency. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount.
Invitrogen lipofectamine 2000 transfection reagent. Transfecting plasmid dna into mcf7 cells using lipofectamine 3000 reagent see all transfection protocols. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Transitbrca transfection reagent research reagent products. The protocal is from thermo fisher scientific important guidelines for transfection 1. Transfection of mcf7 cells with lipofectamine 2000 and isolation of genomic dna abstract. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to.
Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Systematic screening of commonly used commercial transfection. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Prior to transfection, cells that had reached 8085% confluence in 6well culture dishes were added to 1500. Hi, im going to be doing some transfections of mcf7 and mdamb231 cells, with a plasmid containg cat, and my gene of interest. Maintain p19 cells undifferentiated in mem with 10% serum 7. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Mcf7 transfection reagent breast cancer, htb22 altogen.
Using lipofectaminez 2000 for transfection provides the following advantages. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Lipofectamine 2000 transfection reagent from invitrogen. Superior performance for co transfection of sirna and plasmid dna. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Transfection of mcf 7 cells with lipofectamine 2000 and isolation of genomic dna abstract. Transfection protocol lipofectamine 3000 transfection reagent. For additional information on scaling your transfection reaction, see page 4. The following procedure is for simultaneous transfection and plating of raw 264. Does anyone have the efficient protocol to transfect hct116 and mcf7 cells using lipofectamine 2k. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Transfection of human breast adenocarcinoma mcf 7 cells with fugene hd, lipofectamine 2000 and viromer red viromer outperforms major competitors good cell viability vr01lb00, vr01lb01 mcf7. Transfection protocol lipofectamine 3000 transfection.
Highest transfection efficiency in many cell types and formats e. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Have you thought of trying a reverse transfection protocol. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well.
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