Dna transfection lipofectamine 2000 manual

Lipofectamine 2000 transfection reagent dartmouth college. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent. 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. Check the cells 12 days after transfection to determine what ratio gives the highest percentage of gfp positive cells. After incubation, remove media and replace with fresh media. Lipofectamine 2000 transfection protocol 10cm plates reagents. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine.

Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Make sure that cells are greater than 90% confluent and vary dna. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Highest transfection efficiency in many cell types and formats e.

I would start with the smallest amount of lipofectamine recommended 0. I am about to start my stable transfections with lipofectamine 2000. It is best to use polypropylene instead of polystyrene tubes. Plate cells so they will be 7090% confluent at the time of transfection. Lipofection, also known as lipid transfection or liposomebased transfection, uses a lipid complex to deliver dna to cells. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Do any one has protocol for stable transfection with lipofectamine 2000. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Optimized conditions for successful transfection of human. For initial optimization, prepare complexes using a lipofectamine 2000 in microliters to dna in micrograms ratio of 2. Cells were transfected with a firefly luciferase encoding plasmid dna 0. It is not necessary to remove the complexes or change the medium. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.

Using lipofectamine 2000 cd reagent for transfection provides the following advantages. This protocol is for transfecting plasmid dna into mammalian cells. I want to try to use hela cell and lipofectamine 2000 to. Increase transfection efficiency and cell viability with. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. 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. Scaling up or down lipofectamine 3000 reagent transfections. Protocol for a375 cell transfection by fugene hd with.

Transfection efficiencies lower than 50% were not reported in this table. The reagent provides high transfection efficiency in many cells types and formats. Add the lipofectaminedna mixture to each well and gently swirl to mix. Lipofectamine 2000 for transfection provides the following advantages.

Versatileone reagent for dna, rna, and cotransfection efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. On the day of transfection just prior to preparing complexes, plate 1. Lipofectamine 2000 is useful for transfecting dna plasmid. How does transfection with lipofectamine 2000 work. Cells for transfection were harvested at approximately 1,000,000 cells ml. Optimization may be necessary see optimizing plasmid dna transfection. Comparative transfection efficiency of jetprime versus its main competitor. Lipofectamine transfection of c2c12 cells bridges lab. The complex was added to hek293 cells around 7080% confluent in 35mm dish and incubated for 8 h. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent.

All amounts and volumes are given on a per well basis. 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. Dnalipofectamine 2000 cd complexes can be added directly to cells in culture medium. Transfection guide overview of transfection methods. 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. After 5 min, diluted dna was added to diluted lipofectamine 2000 and incubated for 15 min. Take care to maintain a standard seeding protocol between experiments. Exceptional high titers of lentivirus generated from 293t cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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. I previously believed that the best method was using lipofectamine 2000 with plus reagent and an overnight incubation with optimem.

Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Each reagent was tested with a range of transfection reagent. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogen lipofectamine 2000 transfection reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. For additional information on scaling your transfection reaction, see page 4.

Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Consider testing more or less dna while scaling the amount of transitlt1 transfection reagent accordingly. Invitrogen lipofectamine 3000 transfection reagent l3000008.

Highest transfection efficiency in many cell types and formats. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. To transfect cells in different tissue culture formats, vary the. Refer to the cell lines database for a list of cell types successfully. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.

As always, use good cell culture practices and wear your personal protective. Description lipofectamine2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells. I want to try to use hela cell and lipofectamine 2000 to do. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Once a batch of pei is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Lipids are a broad class of fatsoluble biomolecules, such as fats, oils, and waxes. Lipofectamine 2000 transfection reagent from invitrogen. Dilute dna in 250 l of optimem i reduced serum medium without serum or other medium without serum. Description lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. What percentage confluency should be used for stable transfection. Transfection of mcf7 with lipofectamine 2000 maartje vogel. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity.

Following 48 h posttransfection, only a small percentage of hl60 cells cultured in suspension were positive for egfp. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies. I have been doing research with primary rat cardiomyocytes for more than seven years, and plasmid transfection of these cells is not easy. Click here to view protocol gfp expression cell morphology reagent dna. Max % of positive cells after transfection of a gfpplasmid 3. Cells were seeded in 96well flat bottom nontissue culture plate just before transfection with the density 50,000 cells per well in 100. We also provide some tips for optimizing transfection conditions in cultured cells.

Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and. Choosing the right transfection reagent for optimal efficiency. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Three dnas cotransfected with genjet plus in vitro dna transfection reagent left panel in comparison of lipofectamine ltx right panel.

Nucleic acidlipofectamine2000 complexes can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. The cell membranes of animal cells are composed of a bilayer of phospholipids with hydrophilic surfaces facing the cytoplasm and extracellular environment figure 11. Dilute dna in 250 l of optimem i reduced serum medium without serum. Invitrogen lipofectamine 2000 cd transfection reagent 1ml. Lowquality plasmid dna use highly purified, sterile, endotoxin and contaminantfree dna for transfection. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection.

Dna transfection mediated by cationic lipid reagents. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Dnalipofectamine 2000 complexes can be added directly to cells in culture medium. Lipofectamine 2000 dna transfection reagent protocol. Invitrogen lipofectamine 3000 transfection reagent. Incubate the cells at 37c in a co 2 incubator for 2472 hours until they are ready to assay for transgene expression. In this study, lipofectamine reagent and a plasmid with a universal cmv promoter were preferred owing to a relatively high efficiency reported with leukaemia cells in previous studies uchida et al. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows.