Oct 07, 2015 when the cells reached the appropriate confluency 7080%, different concentrations of linearized plasmid, f12 medium and lipofectamine 2000 lifescience were mixed according to the manufacturers protocol. Invitrogen lipofectamine transfection reagent protocols have been. Enhanced transfection efficiency using lipofectamine 3000. Other options will be lipofectamine 2000 invitrogen, transfectam promega. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Have you optimized the transfection condition with regard to 1 dnalipid ratios, 2 cell density, and 3 dna dosages. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. For researchers interested in other delivery methods, we also offer additional invitrogen lipofectamine reagents for mrna transfection and lentiviral production as well as the invitrogen neon transfection system for. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Using lipofectaminez 2000 for transfection provides the following advantages.
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. Evaluating electroporation and lipofectamine approaches. It is possible to plate and transfect the cells on the same day, but cells must adhere to. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. The trusted, simple, broadspectrum transfection reagent for most cell lines. I am trying to do transient transfection, i used fugene 6, which gives me a poor efficiency. Max % of positive cells after transfection of a gfpplasmid 3. C seeding to 60 % confluency in 12 wells 2 hrs prior to transfection medium dmem incl. 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. Mef, mouse embryonal fibroblast cells, plasmid dna, lipofectamine 3000. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 2000 transfection reagent. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofection has become more common than the capo 4 precipitation, because it is just as simple to use but frequently provides a much better transfection efficiency. 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. For the co transfection ofmir16,lna16, antimir16 or scrambled mirna was added with the reporter vectors, 50n m of each mirna or 50nm of each inhibitor or control was transfected using lipofectamine 2000 invitrogen, cergy pontoise, france. For the cotransfection ofmir16,lna16, antimir16 or scrambled mirna was added with the reporter vectors, 50n m of each mirna or 50nm of each inhibitor or control was transfected using lipofectamine 2000 invitrogen, cergy pontoise, france. Transfection was measured by luciferase activity using a conventional assay. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Lipofectamine 2000 reagent thermo fisher scientific. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Cellspecific transfection protocols thermo fisher scientific us. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. I tried to transfect mef with lipofectamine 2000 but i obtained low efficiency of transfection and high mortality. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25.
I use 24well plates for transfections in triplicates. Cell imaging preparation general procedure for mef cells. To investigate the influence of medium and transfection time, primary hepatocytes were transfected with metafecte pro, lipofectamine 2000 or targefect in the presence of dmem figure 2a or optimem, withwithout virofect enhancer, for 3 hours or 16 hours figure 2a, 2b, 2c. Lipofectamine transfection of c2c12 cells bridges lab. Comparative nucleic acid transfection efficacy in primary. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
Mef cells transfected with a luciferase expression plasmid using transit2020. Lipofectamine 3000 transfection reagent the preferred choice for transfection of two types of stem cells. Application note gene delivery solutions stem cell. The presence of optimem resulted in significantly higher transfection rates when lipofectamine 2000 was used. However, you still need to do an optimization on these reagents. Highest transfection efficiency in many cell types and formats e. Transfection of immortalized mefs emory university. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. I tried lipofectamine 2000 and attractene which both had a bad efficiency. Plates were incubated for 30 min over identical magnetic arrays that were either stationary static or moving laterally between 1 and 3 hz at 0. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Mouse embryonic fibroblasts mefs were obtained from the fetuses of a.
Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Despite the importance of transfection in mef cells, there is limited. Divide the cells 1824 hours before transfection to ensure that the cells are actively dividing and reach the appropriate cell density at the time of transfection. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Delivery of episomal vectors into primary cells by means of.
I am trying to transfect mef cells with high transfection efficiency. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. A sample protocol is listed here for transfection experiments performed in 6well plates. For additional information on scaling your transfection reaction, see page 4.
Lipofectaminea 3000 transfection reagent delivers better transfection performance than lipofectaminea 2000 reagent as demonstrated by a customer rui eduardo castro, phd, university of lisbon. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Genscript recommends using lipofectamine 2000 for all transfections. Do transfection in the morning following the protocol.
When the cells reached the appropriate confluency 7080%, different concentrations of linearized plasmid, f12 medium and lipofectamine 2000. Invitrogen lipofectamine 2000 transfection reagent life. Despite this, a satisfactory mechanismbased explanation of their. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. It is a relatively modern method first described by felgner et al. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Transitlt1 is a low toxicity, serumcompatible transfection reagent that eliminates the need for any culture. Cleavage efficiency for geneart talens and crisprs. Lipofectamine 2000 transfection protocol 10cm plates reagents. Hypoxiamicrorna16 downregulation induces vegf expression in. Invitrogen lipofectamine 2000 transfection reagent 15ml.
Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. The transfection efficiencies of the pte cells were 5% effectene. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 invitrogen, paisley, uk and complexes were prepared following the manufacturers recommended protocol. Comparative transfection efficiency of jetprime versus its main competitor. Cytotoxicity and transfection efficiency of lipofectamine 2000 as a. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
The prepared solution was added to the cells, after which cells were incubated at 37. Lipofectamine hescs were plated onto matrigel and mouse embryonic fibroblast mef, at 50% conflu ency, while hff cells were seeded on coated gelatin 0. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Only low transfection rates with lipofectamine, fugene 6 and dotap ca. Optimized conditions for successful transfection of human. At the indicated concentrations of sirna and xtremegene sirna transfection reagent, we observed a greater than 70% depletion of protein x or y relative to the. The suggested amount of dna for mef cells when it is approximately 80% confluent. This protocol is for transfecting plasmid dna into mammalian cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 based transfection 1 seed or grow mef cells to 90% confluence e. The highest transfection efficiency in many cell types and formats e. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Transient transfection of mefs transfection and transduction. Lipofectamine 2000 transfection reagent from invitrogen. Evaluating electroporation and lipofectamine approaches for. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Can anyone recommend a transfection reagent or method for.
The intracellular trafficking mechanism of lipofectamine. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Cells in a 6 well dish, plated and at 70% confluence.
Lipofectamine 2000 thermo fisher scientific and used 2 d posttransfection. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Apr 30, 2007 transient transfection of mefs apr302007 hi, my mef cells are immortalized by sv40 transformation. 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. Improved transfection of huvec and mef cells using dna. Lipofectamine 2000 transfection reagent dartmouth college. How does transfection with lipofectamine 2000 work.
Mcf10a, wi38, huh7, mdamb468, nih3t3, and mef were analyzed by western blot after transfection with sirna targeting gene x or y using the xtremegene sirna transfection reagent. The mechanism of lipofectamine 2000 mediated transmembrane. Use highly purified, sterile, and contaminantfree dna for transfection. Invitrogen lipofectamine 2000 transfection reagent fisher. Comparative analysis of nonviral transfection methods in mouse. Stealthsirna transfection protocol lipofectamine 2000. Plasmid dna preps that are endotoxinfree and have a 260280. 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.
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