Cells in a 6 well dish, plated and at 70% confluence. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. Green fluorescent protein expression was studied under a reverse. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Dna and lipofectamine 2000 were diluted in 100ul optimem gibco each, incubated for 5min, mixed and further incubated for 20min. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Reverse transfection of stealth rnai or sirna using. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfecting sirna using lipofectamine rnaimax pin. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Comparative study of metafecten pro and lipofectamine. Rnaimax reverse transfections lipofectamine thermo.
Cationic lipids, including lipofectamine 2000, are thought to be cytotoxic because they inhibit important proteins like protein kinase c,34 so controlling the amount of lipofectamine 2000 in the transfection formulation is important for achieving satisfactory cytocompatibility. Has anyone here attempted reverse transfection using lipofectamine 3000 reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. 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 and genome engineering fisher scientific. Forward and reverse transfection protocols mirus bio. Maintain p19 cells undifferentiated in mem with 10% serum 7. Optimized delivery of sirna into 3d tumor spheroid. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Reverse transfection an overview sciencedirect topics. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Comparison of reverse transfection efficiency of lipofectamin 2000 with. Invitrogen lipofectamine 2000 transfection reagent life. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. 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. Anyone else have toxicity issues with lipofectamine 2000. We have found that effectene, lipofectamine 2000 and fugene 6 work with this protocol.
To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. Follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells. A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. New reverse genetics and transfection methods to rescue. A sample protocol is listed here for transfection experiments performed in 6well plates. Higher densities can lead to spontaneous differentiation.
Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. 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. A25640 through highly efficient transfection, and ultimately. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. In modified reverse transfections, cells are passaged and plated immediately before transfection complexes are added to the cells. Reverse transfection is possible but it is recommended to use the 2. Genscript recommends using lipofectamine 2000 for all transfections. Pdf optimization of sw480 colon cancer cells transfection with. Lipofectamine 3000 transfection reagent the preferred choice for transfection of two types of stem cells. Transfection protocol lipofectamine 3000 transfection reagent mor. Lipofectamine rnaimax reagent thermo fisher scientific. Can anyone recommend a protocol for transfection of hepg2. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from. Cells were transfected with a firefly luciferase encoding plasmid dna 0.
Evaluation of transfection methods for transient gene. In short, a different rnai molecule is put in each well prior to transfection and combined with diluted lipofectamine 2000 to form complexes in each well. Also, is the nucleic acid in the media responsible for cellcell clumping. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. Reverse primer use the reverse complement sequence of the gene of interest. If so, how could i prevent it from happening when doing a reverse transfection. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates.
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 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Reverse transfection is a technique for the transfer of genetic material into cells. Reverse transfection cell microarray rtcm is a good method for parallel high. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. Cy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection. Add 6l of lipofectamine2000 from biostores and 100 l of. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
For researchers interested in other delivery methods, we also offer additional invitrogen lipofectamine reagents for mrna transfection and lentiviral production as well as the invitrogen neon transfection system for. Pdf optimization of sw480 colon cancer cells transfection. 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. Transfection protocol lipofectamine 3000 transfection reagent gct. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection.
In tube a place 2 g of dna in optimemi for a final volume of 200 l. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine transfection of c2c12 cells bridges lab. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Invitrogen lipofectamine 3000 transfection reagent l3000008. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. Comparative study of metafecten pro and lipofectamine 2000 by reverse transfection cell microarray rtcm introduction reverse transfection cell imcroarray rtcm is a good method for parallel high throughput analyzes of gene function inammalian m cells. Superior performance for cotransfection of sirna and plasmid dna. The protocal is from thermo fisher scientific important guidelines for transfection 1. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax.
Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward trans. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transfection protocol transfection components component cat. Luciferase activity was measured after 24 hours figures 24. Transfection efficiency in a cancer cell line panel.
Hela cells were reverse transfected in 12well plates using 800ng plasmid dna and 2ul lipofectamine 2000 invitrogen. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Reverse transfection magnetofectamine protocol v 1. There are also a few variations of standard reverse transfection protocols that are employed by researchers.
Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Refer to the cell lines database for a list of cell types successfully. Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well.
This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Primer characteristics genome editing applications lipofectamine messengermax reagent increases the likelihood of successful cleavage and recombination with geneart crispr nuclease mrna cat. Highest transfection efficiency in many cell types and formats e.
Invitrogen lipofectamine 2000 transfection reagent. An invitrogen guide mentioning lipofectamine 2000 suggests using reverse transfectin for stealth rnai or rnai. The transfection complex was then added to each well before adding 10 5 hela cells. The method illustrated is the gelatindna method of the reverse transfection approach. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. Rnaimax reverse transfections lipofectamine thermo fisher. Comparative study of metafecten pro and lipofectamine 2000 by. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
Prewarm 50ml of optimum stored in cold room at 4c 10min. Invitrogen lipofectamine 2000 transfection reagent 15ml. Transfection efficiency depends a lot on cell type, though, so you may want to. Induction of osteogenic differentiation of stem cells via. 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. New reverse genetics and transfection methods to rescue arboviruses in mosquito cells. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec commercial transfection reagents in different combinations. Application note gene delivery solutions stem cell. For reverse transfection, cells in optimal growth phase are prepared so that it is possible to seed 2.
Lipofectamine 2000 reagent thermo fisher scientific. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. I have tried optimising the transfections but still end up killing around 50% of my cells. Version 1, 41 8 during the incubation of the printed slides with the transfection reagent, harvest hek293t cells and suspend 10 x 106 cells in 25 ml medium dmem with 10% ifs, 50 unitsml penicillin and 50 gml streptomycin. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. As dna is printed on a glass slide for the transfection process the deliberate introduction of nucleic acids into cells to occur before the addition of adherent cells, the order of addition of dna and adherent cells is reverse that of conventional transfection. Has anyone here attempted reverse transfection using.
Lipofectamine 2000 transfection protocol 10cm plates reagents. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Print bookmark share the next generation in lipid technology pdf 4kb english format file size language. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. A b 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. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol.