Lipofectamine 2000 dna transfection reagent protocol. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 transfection protocol 10cm plates reagents. Therefore, our results suggest that a much higher transfection and viral transduction efficiency can be attained by using lipofectamine 3000 transfection reagent. Plate cells so they will be 7090% confluent at the time of transfection.
A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. It is essential to thoroughly vortex the lipofectamine transfection reagent before use to resuspend lipids that settle during storage. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Request pdf advanced transfection with lipofectamine 2000 reagent. Invitrogen lipofectamine 2000 transfection reagent fisher. The microscopic analysis was performed using antirigi mab. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine 2000 transfection reagent dartmouth college. After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format.
Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Invitrogen lipofectamine 2000 transfection reagent 1. Optimizing transfection lipofectamine 2000 cd reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Visit our product page for additional information and protocols. Transitlt1 is a low toxicity, serumcompatible transfection reagent that eliminates the need for any culture medium change. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Systematic screening of commonly used commercial transfection. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Higher densities can lead to spontaneous differentiation. Transfection efficiency comparison in hela cells using transfectamine. 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.
Primary neurons, sirna, and highthroughput applications lipofectamine 2000 is a. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. 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. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. Please check vansteensel for updated versions of this protocol. Lipofectamine 2000 transfection reagent is easy to usesimply mix with nucleic acid and add to cell culture. Evaluation of transfection methods for transient gene. Start any new transfection by testing the recommended two concentrations of lipofectamine 3000 reagent to determine an optimum amount. Highest transfection efficiency in many cell types and formats e. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many. Lipofectamine 2000 reagent thermo fisher scientific. Please check for updated versions of this protocol.
The intracellular trafficking mechanism of lipofectamine. 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 additional information, and protocols on transfecting suspension cells refer to the manual at. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The reagent provides high transfection efficiency in many cells types and formats. Original article an improved method for increasing the efficiency of. Prewarm 50ml of optimum stored in cold room at 4c 10min. I use 24well plates for transfections in triplicates. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
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. 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. Lipofectamine 3000 transfection reagent hepg2 liver. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. The media needs to be changed at least 1hr prior to transfection. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Transfection protocol lipofectamine 3000 transfection. Serumfree medium is necessary for successful dnalipofectamine 2000. Original article an improved method for increasing the.
Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Add 6l of lipofectamine2000 from biostores and 100 l of. Ideal for highthroughput work simplicity and speed combined with high transfection efficiency make lipofectamine 2000 transfection reagent ideal for transient protein expression or highthroughput rnai transfections. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Viafect transfection reagent transfection assay e4981.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Jun 01, 2004 read advanced transfection with lipofectamine 2000 reagent. Pdf optimization of sw480 colon cancer cells transfection. 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 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein compared to the endogenous pool, as determined by. Although the transfection efficacy and toxicity of transfection reagents. Highest transfection efficiency in many cell types and formats. Each cell line was transfected with a luciferase reporter plasmid using either viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l 50ng of dna or 10l 100ng of dna of the transfection complex in each well of a 96. For your convenience, the essential components of this protocol are now. Lipofectamine 2000 and lipofectamine rnaimax transfection. The trusted, simple, broadspectrum transfection reagent for most cell lines. Plate cells so they will be 6080% confluent at the time of transfection. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells.
Lipofectamine 2000 cd reagent complexes can be added. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Genscript recommends using lipofectamine 2000 for all transfections. 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. Dnaintm transfection reagent family a comparative study of transfection reagents. A sample protocol is listed here for transfection experiments performed in 6well plates. Invitrogen lipofectamine 2000 transfection reagent. Read advanced transfection with lipofectamine 2000 reagent. Caco2 transfection reagent colon carcinoma cells altogen. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. Maintain p19 cells undifferentiated in mem with 10% serum. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas.
Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. 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. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. Transfection protocol lipofectamine 3000 transfection reagent. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. 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. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Advanced transfection with lipofectamine 2000 reagent. The transfection efficacy of lipofectamine 2000 was compromised by its high.
Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with. Invitrogen lipofectamine 2000 transfection reagent 0. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents.
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