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