Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and wait for 5 min at room temperature. Optimized conditions for successful transfection of human. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2. Transient plasmid dna transfection into hela and hacat cells. Outline of protocol steps for the use of lipofectamine 2000 reagent. Invitrogen lipofectamine 2000 transfection reagent. The transfection efficacy of lipofectamine 2000 was compromised by. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine transfection of c2c12 cells bridges lab. An electroporation protocol for efficient dna transfection in pc12 cells. This protocol walks you through our two different means of transfecting mefs and uses for crispr, immunofluorescence, live cell imaging, or other procedures. Invitrogen lipofectamine 2000 transfection reagent 0.
Jun 01, 2000 cationic liposomemediated transfection lipofection has been used as an efficient method for direct gene transfer both in vitro and in vivo. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Despite this, a satisfactory mechanismbased explanation of their. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Its less toxic for cells treatetd with lipo 2000 when you use high cell density 90%. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Select the part name and then you can download the. I want to try to use hela cell and lipofectamine 2000 to.
Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. For additional information on scaling your transfection reaction, see. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. The variables to test include the cell seed density, plasmid amount, and lipofectamine reagent amount, using the lipofectamine protocol as a guide for selecting values for these parameters. Lipofectamine 2000 is a transfection reagent with liposome structure. As for viral production, toxicity will negatively impact the viral yield. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. The reagent provides high transfection efficiency in many cells types and formats. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture.
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 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection reagent from invitrogen. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Cationic liposome and plasmid dna complexes formed in serum. Overwhelmingly more publications cite the use of lipofectamine 2000 transfection. 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. The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. The intracellular trafficking mechanism of lipofectamine. The ratio of transfection reagent to episomal vectors was varied, and the subsequent. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol.
Effectene hela seed 4 x 105 cells the day before transfection and harvest cells 3648 h posttransfection. Invitrogen lipofectamine 2000 transfection reagent fisher. Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid dna is a hydrophilic molecule with a highly negative surface charge due to the. In biontex experiments gfp positivity increased to 90. For reverse transfection b, a549 cells were plated after adding complexes following the protocol below.
Lipofectamine contains lipid subunits that can form. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Pdf optimization of kyse30 esophagus cancer cell line. 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 transient transfection of cos7 cells to. Product is guaranteed for 6 months from the date of shipment if stored properly. I want to try to use hela cell and lipofectamine 2000 to do. Effi ciency and ofp expression were analyzed 72 hours posttransfection and. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. Adjust the amounts of components according to your tissue culture format. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Prepare 6 well plates by adding bakedsterile 22x22 1. For additional information on scaling your transfection reaction, see page 4. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Invitrogen lipofectamine 2000 transfection reagent life. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Superior performance for cotransfection of sirna and plasmid dna with reliable performance for highthroughput applications.
Cationic liposomemediated transfection lipofection has been used as an efficient method for direct gene transfer both in vitro and in vivo. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Thee two most important factors in choosing transfecting reagent are. Download docx, 33kb pdf file option 2 transfection of immortalized mefs. I use 24well plates for transfections in triplicates. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Thee two most important factors in choosing transfecting. Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Despite having a specific protocol, the volume of this reagent should be optimized for use in different cell lines. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Transient plasmid dna transfection into hela and hacat.
Plate cells so they will be 6080% confluent at the time of transfection. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000pgfpn2 transfection. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Advanced transfection with lipofectamine 2000 reagent. Polyethylenimine pei pei qbiogene, morgan irvine, ca and plasmid dna were each diluted with equal volumes of 150 mm nacl. The dnalipofectamine 2000 complexes were then added to each well containing cells and medium. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000, lipofectamine ltx and transitlt1 and of two electroporation systems neon and gene pulser xcell in transiently transfecting undifferentiated pc12 cells. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells important guidelines. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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. Anyone else have toxicity issues with lipofectamine 2000.
Considerations for optimal transfection using lipofectamine 2000 2. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Follow manufacturers handbook protocol unless otherwise indicated. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Highest transfection efficiency in many cell types and formats e. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Invitrogen lipofectamine 2000 transfection reagent 15ml. Lipofectamine transient transfection of cos7 cells to analyze. Plate cells so they will be 7090% confluent at the time of transfection. Make dna lipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. I think for virus production capo4 is milder and efficiently enough. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Intracellular sirna delivery with novel spermine based.
Optimization of transfection methods for huh7 and vero cells. After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. Cationic liposome and plasmid dna complexes formed in. Availability of an efficient transfection protocol is the first determinant in success of gene. An electroporation protocol for efficient dna transfection. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using peyfpn1. This observation is consistent with reports that, regardless of the transfection protocol, delivery of.
Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Delivery of episomal vectors into primary cells by means of. Prewarm 50ml of optimum stored in cold room at 4c 10min. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid dna is a hydrophilic molecule with a highly negative surface charge due to the presence of phosphate groups on. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Lipofectamine 2000 procedure for simultaneously transfecting. Scaling up or down lipofectamine 3000 reagent transfections. Add 6l of lipofectamine2000 from biostores and 100 l of.
Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection protocol 10cm plates reagents. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Troubleshooting for transfection of the crisprcas9 using.
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