Hek293 cells were transiently transfected with lentivirus expression vector plenti cmvgfpdest separately using lipofectamine 3000, 2000 or. Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. The transfection efficacy of lipofectamine 2000 was compromised by. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Highest transfection efficiency in many cell types and formats e. For additional information, and protocols on transfecting suspension cells refer to the manual at. We use it routinely and get 95% transfection efficiency in hek293 cells. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. 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 lipofectamine 3000 transfection reagent. Genetic transfection an overview sciencedirect topics. Lipofectamine 2000 transfection protocol 10cm plates reagents.
Cd 293, 293 sfm ii, and vpsfm are media formulations known to inhibit. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Do not add antibiotics to media during transfection as this causes cell death. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Chemical reagents one of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells was deaedextran vaheri and pagano, 1965. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Systematic screening of commonly used commercial transfection. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. Evaluation of transfection methods for transient gene. Maintain p19 cells undifferentiated in mem with 10% serum 7. Even though cell lines differ in their experimental properties, this protocol will yield efficient transfection at low nano molar concentrations in most of the commonly used cell lines, such as hela, a549, mcf7 or hek293. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 3000 protocol written out with timing.
Other reagents did not demonstrate effective transfection to this cell line. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Youd want to place enough cells to make your transfection well 35%45% confluent since you know that they will double to 70%90% confluent in 24 hours. In addition, high transfection efficiency of hek293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Lipofectamine 2000 for transfection provides the following advantages. Serumfree medium is necessary for successful dnalipofectamine 2000. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Plate cells so they will be 6080% confluent at the time of transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Hek293 cell line origins, cytogenetics, and expression. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. You let them adhere to the surface for an hour or so and then swap the media with fresh media so that they will grow well tip the well to the side and carefully pipette off the medium from.
Transfection protocol lipofectamine 3000 transfection reagent ncih522 lung cancer cells complete growth medium component cat. This protocol isintended for the transfection of adherent mammalian tissue culture 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. Genscript recommends using lipofectamine 2000 for all transfections. Among them, although rnaimax displayed the second highest transfection efficacy, and it had the best safety profile, with 83. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Scaling up or down lipofectamine 3000 reagent transfections. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Universal transfection reagent protocol sigmaaldrich. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 3000 protocol written out with timing specifics. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A guide to transient expression of membrane proteins in.
Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. I have ordered lipofectamine 2000 from thermo fishers scientific. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Hek 293 cells are popular for their ease of growth and transfection hek293 transfection kit, making them a common cell culture in cancer research. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The protocol indicated below is a reverse transfection protocol. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Can anyone provide me with a proper transfection protocol.
For hek293, lipofectamine 3000, fugene and rnaimax showed the highest transfection. An improved method for increasing the efficiency of gene. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Recommended transfection protocols for 24well plate. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. Lipofectamine transfection of c2c12 cells bridges lab. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine plasmid transfection bridges lab protocols. Generally need to have 2 g of dna for each tranfection. In addition, we discuss various transfection agents available from promega as well as general protocols for transfection and specific examples using our transfection reagents. To transfect cells with sirna, follow the protocol as. Sigmas universal transfection reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardtotransfect primary cells.
Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Lipofectamine transfection of c2c12 cells bridges lab protocols. This protocol is for transfecting plasmid dna into mammalian cells. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. For additional information on scaling your transfection reaction, see page 4. Each tube gets 8mls of optimem, 32ug of gag pol, 12. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Original article an improved method for increasing the efficiency of. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. 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.
Optimized conditions for successful transfection of human. The highest transfection efficiency in many cell types and formats e. Lipofectamine rnaimax has a broad peak of activity. Each reagent was used to transfect hek 293, hela, lncap, a549, and. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable. Our results suggest that lipofectamine 3000 outperforms lipofectamine 2000 and fugene hd in transfection efficiency andor protein expression, which is consistent with the claims of the manufacturer who reported it for hek293, hela, lncap, hepg2, and a549 cell lines, the superiority of lipofectamine 3000. 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.
Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Prepare transfection complexes by mixing 40 l of serum. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection.
Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine 2000 reagent thermo fisher scientific. Herein, we report the transfection efficiency and toxicity. Refer to the cell lines database for a list of cell types successfully. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection.
Plate cells so they will be 7090% confluent at the time of transfection. Add 6l of lipofectamine2000 from biostores and 100 l of. Higher densities can lead to spontaneous differentiation. This is necessary because serum is replete with rnases that degrade ribozymes. Lipofectamine 2000 dna transfection reagent protocol.
Lipofectamine 3000, rnaimax, fugene and lipofectamine 2000 all displayed high transfection efficacy 97. I use 24well plates for transfections in triplicates. 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. Hek293 transfection reagent epithelial kidney cells.
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