Invitrogen lipofectamine transfection reagent protocols have been. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Comparative transfection efficiency of jetprime versus its main competitor. Highest transfection efficiency in many cell types and formats e. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. It is a relatively modern method first described by felgner et al.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Mef cells transfected with a luciferase expression plasmid using transit2020. How does transfection with lipofectamine 2000 work. B hela cells were transfected with luciferase encoding plasmid dna using either transit. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. 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.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. The reagent provides high transfection efficiency in many cells types and formats. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Mouse embryonic fibroblasts mefs were obtained from the fetuses of a. Add 6l of lipofectamine2000 from biostores and 100 l of.
The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. I use 24well plates for transfections in triplicates. Lipofectamine 2000 thermo fisher scientific and used 2 d posttransfection. Apr 30, 2007 transient transfection of mefs apr302007 hi, my mef cells are immortalized by sv40 transformation.
I would like to transfect 293t cells with lipofectamine 3000. Optimized conditions for successful transfection of human. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. Invitrogen lipofectamine transfection reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Optimization of transfection methods for huh7 and vero cells.
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. Lipofectamine 2000 transfection reagent dartmouth college. If you find this doesnt work for your specific cell type, then you can look to our cellspecific protocols below for further optimization. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Improved transfection of huvec and mef cells using dna. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in hek293 cells figure 2. However, you still need to do an optimization on these reagents. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The intracellular trafficking mechanism of lipofectamine.
We compared the optimized nucleofection method with other popular nonviral transfection techniques, namely fugene 6 and lipofectamine 2000 using pcs2gfp, ph2bgfp. This protocol is for transfecting plasmid dna into mammalian cells. Pdf file option 2 transfection of immortalized mefs. The highest transfection efficiency in many cell types and formats e. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Plate mefs in wells of a 6well dish so that they are 7080% confluent the next day.
Nanomagnetic gene transfection for nonviral gene delivery in. Cytotoxicity and transfection efficiency of lipofectamine 2000 as a. Transfection was measured by luciferase activity using a conventional assay. Invitrogen lipofectamine 2000 transfection reagent life. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The transfection efficiencies of the pte cells were 5% effectene. Lipofectamine 2000 transfection protocol 10cm plates reagents. This protocol walks you through our two different means of transfecting mefs and uses for crispr, immunofluorescence, live cell imaging, or other procedures.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. The trusted, simple, broadspectrum transfection reagent for most cell lines. Using lipofectaminez 2000 for transfection provides the following advantages. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 dna transfection reagent protocol. Despite the importance of transfection in mef cells, there is limited.
I tried lipofectamine 2000 and attractene which both had a bad efficiency. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Transfection of immortalized mefs emory university. The objective of this work was to examine the potential of oscillating nanomagnetic gene transfection systems magnefectnano for improving the transfection efficiency of nih3t3 mouse embryonic fibroblasts mefs in comparison to other nonviral transfection techniquesstatic magnetofection and the cationic lipid agent, lipofectamine 2000. Maintain p19 cells undifferentiated in mem with 10% serum 7. Efficient transfection of primary zebrafish fibroblasts by. Genscript recommends using lipofectamine 2000 for all transfections.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. I am trying to do transient transfection, i used fugene 6, which gives me a poor efficiency. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Prewarm 50ml of optimum stored in cold room at 4c 10min. Invitrogen lipofectamine 2000 transfection reagent fisher. The firstgeneration lipofection was a singlecomponent system, in which. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0.
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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Cellspecific transfection protocols thermo fisher scientific us. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Other options will be lipofectamine 2000 invitrogen, transfectam promega. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. For additional information on scaling your transfection reaction, see page 4. A sample protocol is listed here for transfection experiments performed in 6well plates. Plate cells so they will be 7090% confluent at the time of transfection.
This is often the best place to start, especially in a new cell line. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. Optimization of transfection methods for huh7 and vero. Lipofectamine transfection of c2c12 cells bridges lab. Date 20 july 2012 description lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Enable high transfection efficiency in novel genome editing applications. Delivery of episomal vectors into primary cells by means of. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. It is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
Max % of positive cells after transfection of a gfpplasmid 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. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells.
When zef1 cells were transfected with fugene 6, the maximal uptake of gfp plasmid was achieved with a 3. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Can anyone recommend a transfection reagent or method for. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Transient transfection of mefs transfection and transduction. Comparative analysis of nonviral transfection methods in mouse. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. 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. Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type.
The mechanism of lipofectamine 2000 mediated transmembrane. Lipofection is most widely used method for transfecting neurons for livecell imaging experiments. Invitrogen lipofectamine 2000 transfection reagent 15ml. I am trying to transfect mef cells with high transfection efficiency.
Lipofectamine 2000 invitrogen, paisley, uk and complexes were prepared following the manufacturers recommended protocol. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Refer to the cell lines database for a list of cell types successfully. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. 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. Despite this, a satisfactory mechanismbased explanation of their.
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. Invitrogen lipofectamine 3000 transfection reagent. Higher densities can lead to spontaneous differentiation. Invitrogen lipofectamine 2000 transfection reagent. Make sure that cells are greater than 90% confluent and vary dna. 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. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. 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. Jurkat cells appear to be less sensitive to toxicity than hek293 and hep g2 cells, as only slight decreases in viability were seen with all transfection reagents in these cells figure 4.