Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Cells that have integrated foreign dna in their genome. Lipofectamine 2000 reagent thermo fisher scientific. For each transfection sample, prepare in separate 1. Cells in a 6 well dish, plated and at 70% confluence. Concentration of plasmid you need for transfection varies depending on the surface area of the tissue culture dish or microplate. Dilute dna plasmids into 500l optimem and lipofectamine2000 in 500l. Transfection efficiencies lower than 50% were not reported in this table. Plate cells so they will be 7090% confluent at the time of transfection. 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 produce a specific protein. Maintain p19 cells undifferentiated in mem with 10% serum 7.
Highest transfection efficiency in many cell types and formats e. Invitrogen lipofectamine 2000 transfection reagent. After 4h aspirate media and refeed cells with normal. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. Mix each tube and let sit for 20 minutes at room temperature. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Cotransfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. Genetic transfection an overview sciencedirect topics. A sample protocol is listed here for transfection experiments performed in 6well plates. Transfection of mcf7 with lipofectamine 2000 maartje vogel.
Cells that have incorporated the foreign dna are called transfectants. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 2000 transfection reagent 1. Transfection protocol lipofectamine 3000 transfection. Liposome lipidbased formulation has the least influence. We recommend gibco optimem i reduced serum medium cat. Rapid isolation of transfectiongrade plasmid dna in high. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection efficiencies decrease with increasing plasmid size and dna lipid ratios should be optimized for each cell line. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Transfection guide overview of transfection methods. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 2000 transfection reagent life technologies, cat. First, hek293 cells were transduced with viral vectors, together with the transfection with plasmids using various tas. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. What is the concentration of plasmids needed for transfection. 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. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine plasmid transfection bridges lab protocols. Evaluation of transfection methods for transient gene. Transfect plasmid dna and sirna at the same time using lipofectamine 2000.
Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Protip endotoxins can inhibit transfection, therefore, plasmid dna purification should include an endotoxin removal step. For the transfection we used the plasmid pmaxgfp transfection control plasmid from amaxa. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Transfection protocol for hpc7 using lipofectamine. I want to try to use hela cell and lipofectamine 2000 to do transfection. Lipofectamin 2000 and large plasmid transfection optimization nov162012 hi all.
Use highquality plasmid dna that is free of proteins, rna, and chemicals for transfections. Your plasmid isnt the problem in terms of size, though the bigger it is, the harder. A few applications that popularly utilize cotransfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. A comparative analysis of akt1 expression in metafectene pro and lipofectamine 2000 transfected cells. I want to try to use hela cell and lipofectamine 2000 to do. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Genscript recommends using lipofectamine 2000 for all transfections. Higher densities can lead to spontaneous differentiation. After 48 h from transfection, cell lysates were prepared. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Cells were transfected with puseakt1 plasmid using metafectene pro or lipofectamine 2000 as described in methods. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Transfection and genome engineering fisher scientific. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. I use 24well plates for transfections in triplicates.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. It was evident that, compared to control plasmid pcdna3, only lipofectaminemediated transfection with plasmid phelper resulted in a 10fold increase in ssaav2mediated transgene expression figure 4a. Add the dna cocktailtransfection buffer mix dropwise to the cells some protocols recommend waiting 1530mins for precipitate to form but i get better transfection when i dont wait 5. On day 1, cotransfect pgipzshrna or plocorf with pcmvdr8. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. It consistently produces high transfection efficiency and high protein overexpression. Some of my cells will dried if i used the volume suggested by the manual. There is a scaling list provided by life technologies for each. Rapid isolation of transfection grade plasmid dna in high.
For high quality plasmid dna, the plasmid should also be propagated in an endonuclease negative e. Lipofectamine 2000 dna transfection reagent protocol. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. We did not find substantial transfection efficiency variations in. Introduction of foreign dna into the nucleus of eukaryotic cells. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Superior performance for cotransfection of sirna and plasmid dna. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Transfection efficiencies were assessed by gfp signal. Lipofectamine 2000 transfection protocol 10cm plates reagents.
Combine the two volumes of optimemdna and optimemlipofectamine. Lipofectamine transfection of c2c12 cells bridges lab. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine 2000 transfection reagent is a proprietary formulation. Use the procedure on below to transfect cells with plasmid dna. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure.
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