Nucleic acid lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The next day the transfection medium was replaced by normal medium. Before you start 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. Advanced transfection with lipofectamine 2000 reagent.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectaminez 2000 for transfection provides the following advantages. Systematic screening of commonly used commercial transfection. Lipofectamine 3000 reagent thermo fisher scientific us. Ddabdope home made and lipofectamine 2000 invitrogen. Transfection guide overview of transfection methods promega. Scaling up or down lipofectamine 3000 reagent transfections. The highest transfection efficiency in many cell types and formats e. For less arsey cell lines, the lipofectamine 2000 is suitable and far cheaper. We are using lipofectamine for transfecting synthetic mirna mimics for the mirna functional analyses and mirna target site validation experiments that we are developing with human cancer cell lines. Highest transfection efficiency in many cell types and formats e. Without changing anything to the transfection conditions you have already setteld, combimag a specific magnetic formulation can be added to complexes.
Jun 29, 2010 ns0 and ag8 cell lines, on the other hand, were efficiently transfected by lipofectamine 2000 and jetpei reagents figure 2. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. It has been shown that the rate of transfection depends on several parameters, including the type of expression vector promoter and enhancer, purity of the expression vectors as well as the transfer method 3. We appreciate greatly the technical service of thermofisher and find them an.
Lipofectamine 3000 reagent provided higher gfp transfection efficiency than. Lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. 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. Aug 17, 2018 transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single cell bioluminescence imaging. It has also been shown to work well with some primary cell lines and is one of the most widely used transfection reagents for use with sirna. Liposomebased dna carriers may induce cellular stress response. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 reagent thermo fisher scientific. The trusted, simple, broadspectrum transfection reagent for most cell lines. Refer to the cell lines database for a list of cell types successfully transfected. Transfections were performed as described in protocol 1, or by replacing the cell culture medium before transfection with serumfree medium and then adding back complete, serumcontaining medium 45 h after the start. Does anyone transfected u251, a172 gbm cell lines with. Gibco highquality selection agents provide unique solutions for your research. Invitrogen lipofectamine 2000 transfection reagent is a proprietary.
Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection. I get consistent results using this lipid for my transfections. Both forward and reverse transfection using transitx2 dynamic delivery system yield superior luciferase expression compared to lipofectamine 2000. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 3000 reagent provided higher gfp transfection ef. A dropdown menu allows you to search the database by cell line, plate type and. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. Jun 16, 2014 stem cell introduces lipofectamine 3000 transfection reagent. Lipofectamine transfection of c2c12 cells bridges lab protocols. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. These mixtures were mixed after 5 minutes at room temperature and incubated for 20 minutes. On the day of transfection just prior to preparing complexes, plate 1. Cells in a 6 well dish, plated and at 70% confluence.
After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. We performed the same experiment using the rat hepatoma fto cell line and observed the same effect of. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 3000 transfection reagent 0. Out of eight different commercially available transfection reagents, there were two possible candidates for use as common chemical transfection reagents, silentfect biorad and hiperfect hts qiagen. Lipofectamine 3000 reagent outperforms lipofectamine 2000 and. Optimemin ul, equal for dna and lipofectamine lipofectamine in ul 25. Im looking to create a database of cell lines i use and their characterisitcs i. Ns0 and ag8 cell lines, on the other hand, were efficiently transfected by lipofectamine 2000 and jetpei reagents figure 2. Fbxo11 targets bcl6 for degradation and is inactivated in. However, of the last 45 years or so, i mostly use effectene from qiagen, high efficiency, low toxicity, need less dna, no need to change medium. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types.
Sydney, australia, lipofectamine 2000 reagent 11668027, invitrogen. Introducing the versatile reagent with the power to. Improved cell viabilitygentle on your cells, with low toxicity. Transfection efficiency and protein expression in various cell lines.
To view a cell lines information, search for it from the front page. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Mix lipofectamine 2000 gently before use, then dilute the appropriate amount in 250. Enhanced transfection efficiency using lipofectamine 3000 reagent. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Versatileone reagent for dna, rna, and cotransfection. Without changing anything to the transfection conditions you have already setteld, combimag a specific magnetic formulation can be added to complexes of lipofectamine 2000 to raise the efficiency. Refer to the cell lines database for a list of cell types successfully. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. 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. Invitrogen lipofectamine ltx reagent with plus reagent. No other plasmid dnaspecific transfection reagent can match the efficiency, convenience, and gentleness of lipofectamine ltx reagent for transfection of primary, challenging, and sensitive cell lines. The transfection activity of lipofectamine 2000 in complete, serum containing media was tested using several common cell lines. Lipofectamine ltx reagent is an advanced solution for gene expression in both common and challenging cell types. Similar effects were seen when using lipofectamine 2000. Invitrogen lipofectamine 3000 transfection reagent from. For example, the gsk cancer cell line genomic profiling database shows that deletions in the fbxo11 locus are present in six of 77 lymphoid cell lines, whereas only three of 255 nonlymphoid cell.
The database can be found in their cell line database. The cell line name and lineage are listed at the top of the cell line page. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension mammalian cell lines compared to similar products. Invitrogen lipofectamine 2000 transfection reagent. Lncap, hepg2, and a549 cell lines in a 96well format, and green. For additional information on scaling your transfection reaction, see page 4. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Other reagents did not demonstrate effective transfection to this cell line. Also, invitrogen has provided a database of different cell lines that have been successfully transfected with lf2000 as well as their other lipid transfection reagents. Sep 25, 2007 lipofectamine 2000 lf2000 is a reagent that i have found fits exactly what i need. The sequencing results were consistent with genbank database. Lipofectamine 2000 procedure for simultaneously transfecting. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent.
Mix gently and incubate for 5 minutes at room temperature. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Fibroblast 3t3, cos7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using peyfpn1. Transfection and genome engineering fisher scientific. Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single cell bioluminescence imaging. Lipo2000 usually works ok for me, not much cell death. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
Dilute sirnachimera with optimem i reduced serum medium invitrogen up to 50 l and vortex for a few seconds dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. But lipo2000 is somewhat better than effectene with some cell lines just a few, but still, e. Methods for sirnamediated reduction of mrna and protein. Transcriptional activity of human acat1 gene p1 promoter and its deletions in diverse cell lines.
Thus, understanding how cells maintain the homeostasis of. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Its transfection efficiency is around 7090% in hepg2 cells as well as vero, hela. Before conducting the transfection analysis in the selected cell lines, the. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. The mutation data table displays a list of mutations specifics to the cell line or gene. I use 24well plates for transfections in triplicates. Silencing of gene expression in cultured cells using small. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. Refer to the transfection collection and the invitrogen transfection guide available at for a list of cell lines and cell types that have been successfully transfected.
I have used lf2000 successfully with the following cell lines. Invitrogen lipofectamine rnaimax transfection reagent. Optimization of sirna transfection for breast and glioma. Is there any software to create a database of cell lines. Optimization of gene transfection in murine myeloma cell. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines.
Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents. Each reagent was used to transfect hek 293, hela, lncap, hepg2, and a549 cell lines in a 96well format, and gfp expression was analyzed 48 hours posttransfection. Cloning and functional analysis of human acyl coenzyme a. Once there, you can do a search for your cell line of interest. For promoter analysis we used the molecular signatures database. Invitrogen lipofectamine 2000 transfection reagent life. About cell stock media, you can use formula as same as normal cells. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine transfection of c2c12 cells bridges lab. Sep, 2005 invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension mammalian cell lines compared to similar products. Anyone else have toxicity issues with lipofectamine 2000.
Molecularcloningofhmenaenahanditssplicevarianthmena. Invitrogen lipofectamine 2000 transfection reagent 1. Enhanced and simple transfection protocol for a variety of cell lines. Lipofectamine plasmid transfection bridges lab protocols. Invitrogen lipofectamine 3000 transfection reagent. Can i use the lipefectamine2000 for mimic mirna transfection. 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.
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