Rnaimax reverse transfections lipofectamine thermo. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 3000 transfection reagent the preferred choice for transfection of two types of stem cells. Pdf optimization of sw480 colon cancer cells transfection. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. Reverse transfection magnetofectamine protocol v 1. Lipofectamine rnaimax reagent thermo fisher scientific.
Follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells. Luciferase activity was measured after 24 hours figures 24. 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. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. The protocal is from thermo fisher scientific important guidelines for transfection 1. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. Prewarm 50ml of optimum stored in cold room at 4c 10min. If so, how could i prevent it from happening when doing a reverse transfection. Transfection efficiency in a cancer cell line panel. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Genscript recommends using lipofectamine 2000 for all transfections. Has anyone here attempted reverse transfection using lipofectamine 3000 reagent.
Invitrogen lipofectamine 3000 transfection reagent l3000008. There are also a few variations of standard reverse transfection protocols that are employed by researchers. Maintain p19 cells undifferentiated in mem with 10% serum 7. Reverse primer use the reverse complement sequence of the gene of interest. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Superior performance for co transfection of sirna and plasmid dna. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Lipofectamine transfection of c2c12 cells bridges lab. 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. Invitrogen lipofectamine 2000 transfection reagent life. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Importantly, the reverse transfection formulations containing a. The transfection complex was then added to each well before adding 10 5 hela cells. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. In modified reverse transfections, cells are passaged and plated immediately before transfection complexes are added to the cells. Has anyone here attempted reverse transfection using. The transfected cell microarray forms in 12 days and is then ready for downstream assays. In tube a place 2 g of dna in optimemi for a final volume of 200 l. New reverse genetics and transfection methods to rescue arboviruses in mosquito cells. Application note gene delivery solutions stem cell.
Transfection protocol lipofectamine 3000 transfection reagent mor. Highest transfection efficiency in many cell types and formats e. Transfecting sirna using lipofectamine rnaimax pin. Reverse transfection is possible but it is recommended to use the 2. 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. Invitrogen lipofectamine 2000 transfection reagent 15ml. Transfection protocol transfection components component cat. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. A sample protocol is listed here for transfection experiments performed in 6well plates. 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. Dna and lipofectamine 2000 were diluted in 100ul optimem gibco each, incubated for 5min, mixed and further incubated for 20min. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. 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.
For reverse transfection, cells in optimal growth phase are prepared so that it is possible to seed 2. The method illustrated is the gelatindna method of the reverse transfection approach. Comparison of reverse transfection efficiency of lipofectamin 2000 with. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. I have tried optimising the transfections but still end up killing around 50% of my cells. An invitrogen guide mentioning lipofectamine 2000 suggests using reverse transfectin for stealth rnai or rnai.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 transfection protocol 10cm plates reagents. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I use 24well plates for transfections in triplicates.
If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. 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. New reverse genetics and transfection methods to rescue. 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. Refer to the cell lines database for a list of cell types successfully. Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Lipofectamine 2000 reagent thermo fisher scientific. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Induction of osteogenic differentiation of stem cells via. 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. 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. 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. 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. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from. Print bookmark share the next generation in lipid technology pdf 4kb english format file size language.
Add 6l of lipofectamine2000 from biostores and 100 l of. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Evaluation of transfection methods for transient gene. 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. 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. Superior performance for cotransfection of sirna and plasmid dna.
Transfection protocol lipofectamine 3000 transfection reagent gct. 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. 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. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Comparative study of metafecten pro and lipofectamine. Cy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax. 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. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec.
The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Lipofectamine 2000 dna transfection reagent protocol. 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. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Transfection efficiency depends a lot on cell type, though, so you may want to. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. We have found that effectene, lipofectamine 2000 and fugene 6 work with this protocol. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount.
Reverse transfection is a technique for the transfer of genetic material into cells. 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. Transfection and genome engineering fisher scientific. Higher densities can lead to spontaneous differentiation. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Pdf optimization of sw480 colon cancer cells transfection with. Anyone else have toxicity issues with lipofectamine 2000. Invitrogen lipofectamine 2000 transfection reagent. Cells in a 6 well dish, plated and at 70% confluence. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward trans.
Reverse transfection cell microarray rtcm is a good method for parallel high. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. Primer characteristics genome editing applications lipofectamine messengermax reagent increases the likelihood of successful cleavage and recombination with geneart crispr nuclease mrna cat. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Reverse transfection of stealth rnai or sirna using lipofectamine. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Hela cells were reverse transfected in 12well plates using 800ng plasmid dna and 2ul lipofectamine 2000 invitrogen. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Also, is the nucleic acid in the media responsible for cellcell clumping.
Can anyone recommend a protocol for transfection of hepg2. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Reverse transfection an overview sciencedirect topics. Reverse transfection of stealth rnai or sirna using. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Forward and reverse transfection protocols mirus bio. Optimized delivery of sirna into 3d tumor spheroid. This protocol is for transfecting plasmid dna into mammalian cells. A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. A25640 through highly efficient transfection, and ultimately.
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