Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine ltx and plus reagent are proprietary formulations for transfecting nucleic acids into a wide range of eukaryotic cells. The protocal is from thermo fisher scientific important guidelines for transfection 1. Mix each tube well by vortexing or flicking the tube. Rnalipofectamine rnaimax complexes must be made in serumfree medium such as optimem. This gives a final volume of 600 l and a final rna concentration of 10 nm. However, the effect of lipofectamine alone on type i ifn response has not been studied in detail. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 3000 reagent protocol lipofectamine 3000. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Evaluation of transfection methods for transient gene. 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.
You can search for similar products in our product directory. 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. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine rnaimax reagent thermo fisher scientific us. Try transfection in 10% dmem or rpmi medium, no need to use serum free medium. Incubate the cells 2448 hours at 37c in a co2 incubator until you are ready to assay for gene knockdown. Invitrogen lipofectamine ltx reagent with plus reagent 0. Roche claims that fugene alters expression of a lot less genes than lipofectamine, so its most likely commercial talk of both companies.
Use it in the same way as lipofectamine 2000, but be sure to use animal origin free reagents. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the or contact technical services for other specialized transfection protocols. Lipofectamine 2000 cd is a 100% synthetic version of lipofectamine 2000. The commercial transfection reagent lipofectamine has been widely used for cytoplasmic delivery of nucleic acids and for cytosolic engagement with intracellular innate immune sensors to trigger type i interferon ifn production. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. Invitrogen lipofectamine ltx reagent with plus reagent. I use 24well plates for transfections in triplicates. Comparison of k2 transfection system with lipofectamine. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Add 6l of lipofectamine2000 from biostores and 100 l of. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Superior transfection reagents we were very happy and surprised to see lipofectamine 3000 reagent provide a more than 10fold difference in transfection ef. Test serum free media formulations for compatibility with lipofectaminez 2000 as some serum free formulations can inhibit cationic lipidmediated transfection. Lipofectin reagent has also been shown to work well, in combination with plus reagent, for transfection of hela cells. Lipofectamine 3000 protocol written out with timing specifics. Dnalipofectamine complexes must be made in serumfree medium such as optimem reduced. Test serumfree media for compatibility with lipofectamine 2000 since some.
Invitrogen lipofectamine 2000 transfection reagent fisher. Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Lipofectamine reagents are widely accepted as goldstandard for the safe. In this paper we characterize the receptors and the signalling pathways involved in the remarkable apoptosis induced by polyi. I would like to ask about the medium in which the transfection should be. The intracellular trafficking mechanism of lipofectaminebased. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000.
Versatileone reagent for dna, rna, and cotransfection. The intracellular trafficking mechanism of lipofectamine. Despite this, a satisfactory mechanismbased explanation of their. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. A new transfection reagent for effective transfection of primary cells, hardtotransfect cells and sensitive established cell lines article pdf available april 2007 with 1,141 reads. Dnalipofectamine ltx and plus complexes must be made in serum free medium such as optimem. Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma.
For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Invitrogen lipofectamine 2000 transfection reagent life. Influence of nodal signalling on pluripotency factor. Plasmid dna and lipofectamine 2000 invitrogen, carlsbad, ca were diluted in two independent 250. Transfection reagent lipofectamine triggers type i interferon. Invitrogen lipofectamine 3000 transfection reagent. For lipofectamine plus transfections, the dna was preincubated with 4. Lipofectamine rnaimax is proprietary animal origin free, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Lipofectamine reagents go online to view related products. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. As a consequence of our measurement protocol, we are able to. How can i optimize the transfection efficiency using lipofectamine.
This is necessary because serum is replete with rnases that degrade ribozymes. I appreciate the fact that the transfection protocol is straightforward, the toxicity appears to be low and we obtained high crispr knockout efficiency. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hardtotransfect cell types. Shipping conditions back to table of content this kit is shipped on wet ice. We have a range of innovative new products available including. As always, use good cell culture practices and wear your personal protective. Invitrogen lipofectamine 3000 transfection reagent 1. Invitrogen lipofectamine 3000 reagent, invitrogen lipofectamine messengermax reagent and more. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. The polycationic compound is preferably polybrene, and the lipid aggregate preferably comprises liposomes of nucleic acid and lipofectamine. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. Invitrogen lipofectamine rnaimax transfection reagent. Enable high transfection efficiency in novel genome editing applications.
Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. 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. Since launching in 1993, invitrogen lipofectamine reagents have become the most referenced transfection reagents with over 50,000 citations to date. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hardtotransfect cell. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Cd 293, 293 sfm ii, and vpsfm are media formulations. Rnalipofectamine rnaimax complexes must be made in serum free medium such as optimem. Lipofectamine reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Prewarm 50ml of optimum stored in cold room at 4c 10min. For additional information on scaling your transfection reaction, see page 4. Invitrogen recommends that antibiotics should not be present in the media during the transfection, as this will cause cell toxicity. Cells are then transfected for 5 h at 37 with fresh serumfree medium containing premixed ribozyme 2.
Therapeutic targeting of atp7b in ovarian carcinoma. You continue to choose these reagents due to their wide acceptance as the best and most reliable available. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, atp7b, using both in vitro and in vivo models. For transfecting dna, rna, and oligonucleotides into mammalian cells, and dna into plant protoplasts. Invitrogen lipofectamine rnaimax transfection reagent 0. Lipofectamine 2000 transfection reagent from invitrogen. Transit2020 transfection reagent research reagent products. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Application note lipofectamine stem transfection reagent versatility and performance of lipofectamine stem transfection reagent introduction the ability of stem cells to selfrenew and differentiate into various specialized cell types promises to contribute greatly to future applications in regenerative medicine and to the. Transfection and genome engineering fisher scientific. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture medium.
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. Invitrogen lipofectamine 2000 transfection reagent. Expression of atp7a and atp7b was examined in ovarian cancer cell lines by realtime reverse. Lipofectamine 2000 from invitrogen biocompare product.
Once reconstituted in nuclease free water, the sirna is ready to transfect at your choice of final concentration. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Testicular germ cell tumours tgcts are characterised by an overall high cisplatinsensitivity which has been linked to their continued expression of pluripotency factors. Invitrogen lipofectamine 3000 transfection reagent fisher. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. Maybe specifiy what cellscell lines your working with, optimal reagents may differ between different cells. Transfection guidelines see page 2 to view guidelines for transfection of sirnas using lipofectamine rnaimax reagent. Life technologies corporation andor its affiliates warrant their products as set forth in the life. Lipofectamine 2000 was simple to use and unlike other reagents displayed very low toxicity. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium. You can generally use other serum free medium instead of optimem.
Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture medium, in the. Prior to transfection, hek 293 cells are preincubated with serum free dmem medium for 1 h. Their free diffusion also decreases by a factor 4 dlfn. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. At 5 h posttransfection, heatinactivated fcs is added to a final concentration of 6%. Invitrogen lipofectamine 2000 transfection reagent 1. Lipofectamine 2000 dna transfection reagent protocol. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform transfection in different cell culture formats, refer to table2 and invitrogen lipofectamine. The protocol supplied by invitrogen was straight forward and also very accurate as we found that deviations from the protocol often resulted in lower transfection efficiencies and higher cell death. The average lentiviral titers in our preparations are around 56x 10 5 x 10. The solutions were combined and mixed by pipetting up and down once. Invitrogen lipofectamine rnaimax transfection reagent offers an advanced, efficient solution for sirna delivery.
Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Lipofectamine reagents offer you the ability to consistently transfect many cell types. A method is provided for enhancing transfection efficiency of eukaryotic cells comprising contacting said cells with a lipid aggregate comprising nucleic acid and a polycationic lipid composition in the presence of a polycationic compound and serum. Transfection protocol transfection components component cat. Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine rnaimax, what medium for transfection. 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. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Us5627159a enhancement of lipid cationic transfections. Invitrogen lipofectamine 3000 transfection reagent from.