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 contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. The trusted, simple, broadspectrum transfection reagent for most cell lines. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. Combine the two solutions, mix gently and incubate for 20 min. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 2000 from invitrogen biocompare product.
After the 5minute incubation, combine the diluted oligomer with the diluted lipofectamine 2000. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Life technologies corporation andor its affiliates warrant their. 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. 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. Lipofectamine 2000 dna transfection reagent protocol. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Invitrogen lipofectamine 3000 transfection reagent.
Versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Optimizing transfection of primary human umbilical vein. Product is guaranteed for 6 months from the date of shipment if. We have compared the efficiency of three different chemical transfection reagents lipofectamine 2000, lipofectamine ltx and transitlt1 and of two electroporation systems neon and gene pulser xcell in transiently transfecting undifferentiated pc12 cells. The intracellular trafficking mechanism of lipofectaminebased. Guides for easy transfection of notsocom mon cell lines are also available at lipofectamine2000. After 5 minute incubation, combine the diluted dna with diluted lipofectamine 2000 cd reagent total volume 100 l. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died.
Optimized conditions for successful transfection of human. Invitrogen lipofectamine 2000 transfection reagent. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Highest transfection efficiency in many cell types and formats e. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced. Lipofectamine 2000 transfection reagent dartmouth college. Mix gently and incubate for 20 minutes at room temperature to allow the dnalipofectamine 2000 complexes to form. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Advanced transfection with lipofectamine 2000 reagent. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format.
According to the manufacturers protocol invitrogen, dh10bac competent cells were transfected with a recombinant pfastbac1, and dna was purified by the suggested miniprep method marligen bioscience. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. The most efficient reagents were the cationic lipid reagents, lipofectamine 2000 and ltx, from invitrogen, which were able to genemodify up to 38% of huvec. 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. An electroporation protocol for efficient dna transfection. Lipofectamine 2000 transfection reagent from invitrogen.
You can search for similar products in our product directory. The intracellular trafficking mechanism of lipofectamine. Does lipofectamine 2000 give a stable transfection. Here we compared the transfection and viral transduction efficiency using. Lipofectamine 2000 is a proprietary formulation for the transfection of. I use 24well plates for transfections in triplicates. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells. These mixtures were mixed after 5 minutes at room temperature and incubated for 20 minutes. Transitlt1 transfection reagent mirus, madison, wi, usa, lipofectamine 2000 and lipofectamine ltx invitrogen. Our lab then bought the hek flpin cells from invitrogen, and i was able to generate the wildtype mrp1 stable hek cell line within roughly a month. Enable high transfection efficiency in novel genome editing applications.
Invitrogen transfection guide available at for a list of cell lines. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage. Anyone else have toxicity issues with lipofectamine 2000. Invitrogen lipofectamine 2000 transfection reagent 1. So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for each well, use that amount for three wells. The next day the transfection medium was replaced by normal medium. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. For using lipofectamine 2000 reagent, the above plasmids were. Despite this, a satisfactory mechanismbased explanation of their. Transfections were performed in 24well plates using 0. Dnalipofectamine 2000 complexes can be added directly to cells in. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Invitrogen 11668019 view more versions of this product.
Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. However, recently i tried to combine overexpression with scratch wound healing assay. An improved method for increasing the efficiency of gene. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. 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. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. Invitrogen lipofectamine 2000 transfection reagent 15ml. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 cd reagent thermo fisher scientific. After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500.