Plate cells so they will be 7090% confluent at the time of transfection. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Superior performance for cotransfection of sirna and plasmid dna. Transient plasmid dna transfection into hela and hacat. Figure 1 illustrates a typical, reagentbased transfection protocol. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. I am using lipofectamine 2000 reagent from life technologies. Lipofectamine transfection of c2c12 cells bridges lab.
This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. A dynamic transfection system for the delivery of plasmid dna, sirnamirna and. This protocol is for transfecting plasmid dna into mammalian cells. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Invitrogen lipofectamine 2000 transfection reagent 15ml. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Test serumfree media formulations for compatibility with lipofectamine. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338 100. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. 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. 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 are the most efficacious and showed little or. The madindarby canine kidney cell line mdck ii was cultured in 75 cm. Scaling up or down lipofectamine 3000 reagent transfections. A549 a or mdck b cells were transfected with luciferase encoding. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market.
The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. For other tissue culture formats, refer to the user protocol pdf. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transient transfection of polarized epithelial monolayers with cftr. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. 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. The development of multiresistant strains of staphylococcus aureus is now a major public health hazard. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression.
Cells should be plated on collagencoated dishes, as for normal propagation. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Invitrogen lipofectamine transfection reagent protocols have been optimized. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. I use 24well plates for transfections in triplicates. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Higher densities can lead to spontaneous differentiation. Invitrogen lipofectamine 3000 transfection reagent l3000008. A highly efficient method of transfecting hep g2 cells. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension.
L lipofectamine 2000 and incubated together with the cells in optimem i medium invitrogen for 4 h. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine 2000 dna transfection reagent protocol. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfection of mdck cells with human siat7e and its effects on cellcell. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Viral stocks are subsequently titered by plaque assay on mdck cells. Transfection of mcf7 with lipofectamine 2000 maartje vogel.
Invitrogen lipofectamine 2000 transfection reagent. Advanced transfection with lipofectamine 2000 reagent. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. 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.
If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Efficient electroporation of dna and protein into confluent and. Transitbrca transfection reagent research reagent products. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. G418, lipofectamine 2000 transfection reagent, trizolls reagent and. Cotransfection protocol for delivery of multiple plasmid dnas in a 6well plate with transitx2. Transfection optimizationhow to get it right biocompare. 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.
Dna complexes were added to cells that had already been allowed to attach to the plate b. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. 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. 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. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Anyone familiar with transfection of mdck epithelial cells.
Highest transfection efficiency in many cell types and formats e. 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. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine 2000 transfection reagent dartmouth college.
Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to. Anyone familiar with transfection of mdck epithelial cells using. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Highest transfection efficiency in cells at 90% confluency. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Cotransfection protocol for multiple plasmid dnas mirus bio. To transfect cells with sirna, follow the protocol as described for dna but. Cellspecific transfection protocols thermo fisher scientific es. For additional information on scaling your transfection reaction, see page 4. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.
Cellspecific transfection protocols thermo fisher scientific us. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. While it delivers efficiently to many breast cancer cell types, it also maintains low cellular toxicity resulting in reduced experimental biases.
Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Transient plasmid dna transfection into hela and hacat cells. Invitrogen lipofectamine 3000 transfection reagent. 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.
Transitx2 for crispr plasmid delivery protocol pdf. Lipofectamine 2000 reagent thermo fisher scientific. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Optimized conditions for successful transfection of human. Do any one has protocol for stable transfection with lipofectamine 2000. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Lipofectamine 2000 transfection reagent from invitrogen.
A sample protocol is listed here for transfection experiments performed in 6well plates. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Anyone familiar with transfection of mdck epithelial cells using paxillin, vinculin, zo1 and ecadherin plasmids. Proceedings of the national academy of sciences sep 2009, 106. The intracellular trafficking mechanism of lipofectamine. Invitrogen transfection reagent, recommended transfection sku, protocol.
Transfection protocol lipofectamine 3000 transfection. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Dnatransfection of madindarby canine kidney cells mdck ii cell. Conversion of mdck cell line to suspension culture by. Huvec transfection kit, umbilical vein endothelial cell. What percentage confluency should be used for stable transfection. Mdck, madindarby canine kidney, sirna, lipofectamine rnaimax, 778075. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.