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. This is necessary because serum is replete with rnases that degrade ribozymes. Transfection protocol lipofectamine 3000 transfection reagent. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 reagent thermo fisher scientific.
Hek293 standard transfection protocol 24well plate. Refer to the cell lines database for a list of cell types successfully. 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. Original article an improved method for increasing the efficiency of. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Prepare transfection complexes by mixing 40 l of serum. Recommended transfection protocols for 24well plate. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 dna transfection reagent protocol.
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. This protocol isintended for the transfection of adherent mammalian tissue culture cells. Can anyone provide me with a proper transfection protocol. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine rnaimax has a broad peak of activity. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Herein, we report the transfection efficiency and toxicity. Lipofectamine 2000 for transfection provides the following advantages. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Systematic screening of commonly used commercial transfection. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25.
Higher densities can lead to spontaneous differentiation. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A sample protocol is listed here for transfection experiments performed in 6well plates. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. 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. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Evaluation of transfection methods for transient gene.
Among them, although rnaimax displayed the second highest transfection efficacy, and it had the best safety profile, with 83. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 3000 protocol written out with timing. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable. Serumfree medium is necessary for successful dnalipofectamine 2000. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Do not add antibiotics to media during transfection as this causes cell death. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Hek293 cells were transiently transfected with lentivirus expression vector plenti cmvgfpdest separately using lipofectamine 3000, 2000 or.
Lipofectamine 2000 transfection protocol 10cm plates reagents. An improved method for increasing the efficiency of gene. 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. Lipofectamine transfection of c2c12 cells bridges lab. A guide to transient expression of membrane proteins in. Hek293 transfection reagent epithelial kidney cells. The transfection efficacy of lipofectamine 2000 was compromised by. Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Plate cells so they will be 7090% confluent at the time of transfection. In addition, we discuss various transfection agents available from promega as well as general protocols for transfection and specific examples using our transfection reagents. Universal transfection reagent protocol sigmaaldrich. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics. Lipofectamine plasmid transfection bridges lab protocols. Other reagents did not demonstrate effective transfection to this cell line.
Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Chemical reagents one of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells was deaedextran vaheri and pagano, 1965. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. For additional information on scaling your transfection reaction, see page 4. Transfection protocol lipofectamine 3000 transfection reagent ncih522 lung cancer cells complete growth medium component cat.
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. In addition, high transfection efficiency of hek293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. I have ordered lipofectamine 2000 from thermo fishers scientific. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. Scaling up or down lipofectamine 3000 reagent transfections. 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. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For additional information, and protocols on transfecting suspension cells refer to the manual at. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. The protocol indicated below is a reverse transfection protocol. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Hek 293 cells are popular for their ease of growth and transfection hek293 transfection kit, making them a common cell culture in cancer research. Hek293 cell line origins, cytogenetics, and expression. Highest transfection efficiency in many cell types and formats e. 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. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine transfection of c2c12 cells bridges lab protocols. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. The highest transfection efficiency in many cell types and formats e. Lipofectamine 3000 protocol written out with timing specifics.
Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. 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. Genetic transfection an overview sciencedirect topics. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Each tube gets 8mls of optimem, 32ug of gag pol, 12. For hek293, lipofectamine 3000, fugene and rnaimax showed the highest transfection. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Lipofectamine 3000, rnaimax, fugene and lipofectamine 2000 all displayed high transfection efficacy 97. I use 24well plates for transfections in triplicates. To transfect cells with sirna, follow the protocol as. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 15630080 1. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media.
Maintain p19 cells undifferentiated in mem with 10% serum 7. Generally need to have 2 g of dna for each tranfection. We use it routinely and get 95% transfection efficiency in hek293 cells. 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. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. 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. Cd 293, 293 sfm ii, and vpsfm are media formulations known to inhibit.
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. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Optimized conditions for successful transfection of human. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. This protocol is for transfecting plasmid dna into mammalian cells. Deaedextran is a cationic polymer that tightly associates. Plate cells so they will be 6080% confluent at the time of transfection. Each reagent was used to transfect hek 293, hela, lncap, a549, and. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0.
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