To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. This reference provides a customer generated protocol for transfecting plasmid dna into primary mouse neural. Reverse transfection of stealth rnai or sirna using. Reverse transfection of plasmid dna automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in highthroughput assays. 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. Reverse transfection of stealth rnai or sirna using lipofectamine. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax. Transfection protocol lipofectamine 3000 transfection reagent mdamb231 breast cancer cells complete growth medium component cat. The media needs to be changed at least 1hr prior to transfection. The mechanism of lipofectamine 2000 mediated transmembrane.
A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. Add 6l of lipofectamine2000 from biostores and 100 l of. Rnaimax reverse transfections lipofectamine thermo fisher. Forward and reverse transfection protocols mirus bio. In the protocol described here, we use a pool of three sirna duplexes per target gene per.
Refer to the cell lines database for a list of cell types successfully. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. This protocol is for use with adherent cells, as transfection efficiency for. How does transfection with lipofectamine 2000 work. Also, is the nucleic acid in the media responsible for cellcell clumping. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Highthroughput applications, such as knockdown studies or target screenings, often include cell transfection. Transfection protocol lipofectamine 3000 transfection. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection.
Mir2304 and lipofectamine 2000 transfection reagent life technologies, cat. Invitrogen lipofectamine 3000 transfection reagent fisher. To perform transfection experiments in other cell culture plates, simply multiply the. Reverse transfection combines rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 transfection protocols. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. Transfection guide overview of transfection methods. 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. I am attempting to deliver a plasmid 9000 bp in size into hardtotransfect lung fibroblasts. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. 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. May 21, 2018 transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best performing polymeric nanoparticles transfected 57.
What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual. Has anyone here attempted reverse transfection using. Transfection guide overview of transfection methods promega. Automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in high. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery.
On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. 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. 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. Optimizing transfections to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rnai duplex and lipofectamine rnaimax concentrations. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Optimized delivery of sirna into 3d tumor spheroid cultures. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Follow these general guidelines when using lipofectamine 2000 to reverse transfect. Enable high transfection efficiency in novel genome editing applications.
Reverse transfection instruction manual the two most powerful transfection technologies in one kit. Transfection reagents product protocols thermo fisher scientific. Reverse transfection protocol reverse transfection combines dna or rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 or magnetofectamine. 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. Rnaimax forward transfections lipofectamine thermo.
Transfection efficiencies lower than 50% were not reported in this table. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. This protocol is based on that from life technologies called. 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. The intracellular trafficking mechanism of lipofectamine. Reverse transfection protocol for dna in 96well plates using transitx2 transfection reagent. Transfection protocol lipofectamine 3000 transfection reagent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection.
Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Cloned genes can be transfected into cells for biochemical characterization. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. It would be great if anyone can share a trusted protocol with me.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward trans. An invitrogen guide mentioning lipofectamine 2000 suggests using reverse transfectin for stealth rnai or rnai. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection protocol lipofectamine 3000 transfection reagent ncih292 lung cancer cells complete growth medium component cat. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best performing polymeric nanoparticles. For additional information on scaling your transfection reaction, see page 4. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent.
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. Has anyone here attempted reverse transfection using lipofectamine 3000 reagent. Reverse transfection on cell arrays for high content. If so, how could i prevent it from happening when doing a reverse transfection. I tried reversetransfecting hepg2 cells using lipofectamine 2000. Transfection protocol lipofectamine 3000 transfection reagent gct. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25.
Lipofectamine 2000 transfection reagent from invitrogen. Akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest such as a luciferase or gfp encoding plasmid into cells is critical prior to high throughout screening. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Gibco rpmi 1640 with glutamax supplement 61870036 10%. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. A sample protocol is listed here for transfection experiments performed in 6well plates. Therefore, we recommend testing different batch numbers of the transfection reagent and. Maintain p19 cells undifferentiated in mem with 10% serum 7. Our lab uses lipofectamine 3000 for transfections, but the only protocols for reverse transfection i can find on thermofisher scientifics website deal with their rnaimax reagent. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. There are also a few variations of standard reverse transfection protocols that are employed by researchers.
Max % of positive cells after transfection of a gfpplasmid 3. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Reverse transfection an overview sciencedirect topics. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. 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. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from invitrogen, with the unique magnetofection based reagent combimag, from oz biosciences list of magnetofectamine kits. Transfection protocol lipofectamine 3000 transfection reagent gct lung cancer cells complete growth medium component cat. Concentration of dna for stable transfection with lipofectamine 2000 and the per transfection and transduction. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Invitrogen lipofectamine 3000 transfection reagent. It is best to use polypropylene instead of polystyrene tubes. Invitrogen lipofectamine 2000 transfection reagent.
For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine is formulated to give high transfection efficiency with little. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. In a reverse transfection protocol, cells are added directly to a plate. Reverse transfection protocol for dna in 96well plates using transit2020 transfection reagent before you start. Rnaimax reverse transfections lipofectamine thermo. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Genscript recommends using lipofectamine 2000 for all transfections. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
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. We have found that effectene, lipofectamine 2000 and fugene 6 work with this. Invitrogen lipofectamine 3000 transfection reagent l3000008. Lipofectamine 2000 reagent thermo fisher scientific.
Prewarm 50ml of optimum stored in cold room at 4c 10min. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Invitrogen lipofectamine 3000 transfection reagent 1. Page 2 gelatin preparation, dna purification and microarray printing page 2 cell preparation page 3 reverse transfection of printed microarrays page 4 detection methods page 5 immunofluorescence page 6 autoradiography page 6 western blots page 6 tips and information page 7 alternative method. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs. We observed a high batchtobatch variation in performance of the lipofectamine 2000 transfection reagent.