Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Transient transfection of polarized epithelial monolayers with cftr. For other tissue culture formats, refer to the user protocol pdf. Invitrogen lipofectamine 3000 transfection reagent. Test serumfree media formulations for compatibility with lipofectamine.
Dnatransfection of madindarby canine kidney cells mdck ii cell. Genscript recommends using lipofectamine 2000 for all transfections. Higher densities can lead to spontaneous differentiation. How does transfection with lipofectamine 2000 work. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. I am using lipofectamine 2000 reagent from life technologies. Cotransfection protocol for multiple plasmid dnas mirus bio. Anyone familiar with transfection of mdck epithelial cells using paxillin, vinculin, zo1 and ecadherin plasmids. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Scaling up or down lipofectamine 3000 reagent transfections.
Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Viral stocks are subsequently titered by plaque assay on mdck cells. Anyone familiar with transfection of mdck epithelial cells. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Invitrogen lipofectamine 2000 transfection reagent. Cellspecific transfection protocols thermo fisher scientific us. Conversion of mdck cell line to suspension culture by. Optimized conditions for successful transfection of human.
Invitrogen lipofectamine 2000 transfection reagent versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines manufacturer. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Figure 1 illustrates a typical, reagentbased transfection protocol. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. For additional information on scaling your transfection reaction, see page 4. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. While it delivers efficiently to many breast cancer cell types, it also maintains low cellular toxicity resulting in reduced experimental biases. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Cotransfection protocol for delivery of multiple plasmid dnas in a 6well plate with transitx2.
Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Highest transfection efficiency in cells at 90% confluency. Highest transfection efficiency in many cell types and formats e. The development of multiresistant strains of staphylococcus aureus is now a major public health hazard. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 are the most efficacious and showed little or. G418, lipofectamine 2000 transfection reagent, trizolls reagent and. Lipofectamine 2000 transfection reagent dartmouth college. The recent emergence of vancomycinresistant strains in multiple countries, including the united states, has further underscored the importance of identifying microbial factors that may lead to the development of preventive vaccines and novel antimicrobial therapy. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection optimizationhow to get it right biocompare. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Mdck, madindarby canine kidney, sirna, lipofectamine rnaimax, 778075. Cells should be plated on collagencoated dishes, as for normal propagation. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. The plasmids were purified using the qiaprep spin miniprep kit qiagen and were used to transfect mdck cells using lipofectamine 2000 reagent under manufacturers protocol invitrogen. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a. Invitrogen lipofectamine transfection reagent protocols have been optimized. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences.
Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338 100. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to. Lipofectamine 2000 dna transfection reagent protocol. 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. L lipofectamine 2000 and incubated together with the cells in optimem i medium invitrogen for 4 h. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Efficient electroporation of dna and protein into confluent and. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Cellspecific transfection protocols thermo fisher scientific es.
Advanced transfection with lipofectamine 2000 reagent. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Superior performance for cotransfection of sirna and plasmid dna. Transient plasmid dna transfection into hela and hacat cells. The reagent provides high transfection efficiency in many cells types and formats. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. A549 a or mdck b cells were transfected with luciferase encoding. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Invitrogen lipofectamine 3000 transfection reagent l3000008.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. I use 24well plates for transfections in triplicates. Transitbrca transfection reagent research reagent products. A dynamic transfection system for the delivery of plasmid dna, sirnamirna and. Invitrogen lipofectamine 2000 transfection reagent 15ml. Transfection protocol lipofectamine 3000 transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transient plasmid dna transfection into hela and hacat. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine transfection of c2c12 cells bridges lab. 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. Anyone familiar with transfection of mdck epithelial cells using.
The madindarby canine kidney cell line mdck ii was cultured in 75 cm. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfection of mdck cells with human siat7e and its effects on cellcell. 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. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. A sample protocol is listed here for transfection experiments performed in 6well plates. Invitrogen transfection reagent, recommended transfection sku, 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 lipofectamine 3000 transfection reagent. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. A highly efficient method of transfecting hep g2 cells. Dna complexes were added to cells that had already been allowed to attach to the plate b.
Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. 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. Transitx2 for crispr plasmid delivery protocol pdf. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Do any one has protocol for stable transfection with lipofectamine 2000. Huvec transfection kit, umbilical vein endothelial cell. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead the cells are dying after incubation with reagent and dna. What percentage confluency should be used for stable transfection. To transfect cells with sirna, follow the protocol as described for dna but.
This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Proceedings of the national academy of sciences sep 2009, 106. Lipofectamine 2000 reagent thermo fisher scientific. Plate cells so they will be 7090% confluent at the time of transfection.
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