Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format using 0. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Does anyone have experience transfecting shsy5y cells with lipofectamine 2000.
Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection efficiency and is a helpful tool for gene expression studies as well as for assessing cellular functionality. Caco2 transfection reagent colon carcinoma cells altogen. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Maintain p19 cells undifferentiated in mem with 10% serum. The protocal is from thermo fisher scientific important guidelines for transfection 1. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. 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. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture. Jun 01, 2004 read advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. A highly efficient method of transfecting hep g2 cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Higher densities can lead to spontaneous differentiation. 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. 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. Dilute lipofectamine 3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated.
Comparative transfection efficiency of jetprime versus its main competitor. Transfection and genome engineering fisher scientific. Lipofectamine 2000 reagent and lipofectamine ltx reagent. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Prewarm 50ml of optimum stored in cold room at 4c 10min. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Please check for updated versions of this protocol. Transfection efficiency in a cancer cell line panel. Can anyone recommend a protocol for transfection of hepg2 cells with sirna and lipofectamine. Lipofectamine 2000 cd reagent complexes can be added. Optimem reduced serum medium, no phenol red life technologies, cat.
Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Transfection protocol 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 invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lipofectamine 3000 transfection reagent protocol transfect cells according to the following table. Does anyone have experience transfecting shsy5y cells. To transfect cells with sirna, follow the protocol as.
Optimized conditions for successful transfection of human. A sample protocol is listed here for transfection experiments performed in 6well plates. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Lipofectamine 2000 transfection reagent life technologies, cat. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1 and with. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. This is most often an endogenous proteincoding gene, although some noncoding rnas can also be silenced effectively. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. For additional information, and protocols on transfecting suspension cells refer to the manual at. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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. Review and cite lipofectamine protocol, troubleshooting and other.
The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Add 6l of lipofectamine2000 from biostores and 100 l of. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Scaling up or down lipofectamine 3000 reagent transfections. 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.
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. You can generally use other serumfree medium instead of optimem. Formulation of lipofectamine 2000 and lipofectin signagen blog. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 transfection protocol 10cm plates reagents. Plate cells so they will be 7090% confluent at the time of transfection. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of.
Invitrogen lipofectamine 2000 transfection reagent. Invitrogen lipofectamine 2000 transfection reagent 0. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine 2000 and lipofectamine rnaimax transfection. For the cell lines easy to transfection, you can use either of the procedures reverse or forward.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Do not add antibiotics to media during transfection as this will cause cell death. I use 24well plates for transfections in triplicates. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon. For the following protocol, we specify volumes for a 12well plate.
Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and dna concentrations, liposomedna. Does anyone have experience transfecting shsy5y cells with. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. It consistently produces high transfection efficiency and high protein overexpression. For additional information on scaling your transfection reaction, see page 4. 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. I want to try to use hela cell and lipofectamine 2000 to do. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine 3000 protocol written out with timing.
Rnai in cultured mammalian cells using synthetic sirnas. Plate cells so they will be 6080% confluent at the time of transfection. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 3000 protocol written out with timing specifics. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Using the transfection protocol outlined above, we routinely achieve transfection. Pool dishes of cells that have been prepared for transfection. Lipofectamine 3000 transfection reagent hepg2 liver. Take care to maintain a standard seeding protocol between experiments because transfection efficiency is sensitive to culture confluence.
Lipofectamine 3000 protocol written out with timing specifics regarding the tran. Hela transfection is usually pretty standard in terms of protocols just make sure your. Use the indicated volume of dna and p3000 reagent with each of the two volumes of lipofectamine. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells.
Lipofectamine 2000 procedure for simultaneously transfecting. Please check vansteensel for updated versions of this protocol. Use the indicated volume of dna and p3000 reagent with each of the two volumes of lipofectamine 3000 when. Prior to use of lipofection on an alternative cell line, we strongly recommend doing several transfections using different concentrations of lipofectamine 2000, as. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Can anyone recommend a protocol for transfection of hepg2. It is worth checking their website before starting, as they have a number of protocols provided for standard cell lines. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection protocol lipofectamine 3000 transfection. Product isguaranteed for 6 months from the date of shipment if stored properly.