Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis. Studies were undertaken to evaluate and compare the transfection efficacy. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. While it delivers efficiently to many breast cancer cell types, it also maintains. Lipofectamine 2000 reagent thermo fisher scientific. 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.
In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I am using lipofectamine 2000 reagent from life technologies. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Transfection protocol of lipofectamine rnaimax can be. Invitrogen lipofectamine 2000 transfection reagent life.
Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Invitrogen lipofectamine 3000 transfection reagent fisher. Efficient electroporation of dna and protein into confluent and. G418, lipofectamine 2000 transfection reagent, trizolls reagent and. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Refer to the cell lines database for a list of cell types successfully. For cellfectin dmriec, and lipofectamine 2000 reagents this is normal. The mechanism of lipofectamine 2000 mediated transmembrane. 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.
Higher densities can lead to spontaneous differentiation. Lipofectamine is formulated to give high transfection efficiency with little. Lipofectamine 2000 dna transfection reagent protocol. The use of these different lipofectamine 2000 amounts for transfection showed. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. Superior performance for co transfection of sirna and plasmid dna. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Prewarm 50ml of optimum stored in cold room at 4c 10min. Welcome to the cheung lab lab protocols lipofectamine. Transitbrca transfection reagent research reagent products. Lipofectamine 2000 are the most efficacious and showed little or. Comparative transfection efficiency of jetprime versus its main competitor.
The development of multiresistant strains of staphylococcus aureus is now a major public health hazard. 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. 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. Transfection optimizationhow to get it right biocompare.
Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Transitbrca transfection reagent is the first dedicated breast cancer cell line specific reagent available to researchers. I use 24well plates for transfections in triplicates. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Invitrogen transfection reagent, recommended transfection sku, protocol. The madindarby canine kidney cell line mdck ii was cultured in 75 cm. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Can anyone please explain why we need the p3000 in addition to lipofectamine or how p3000 helps in adition to the lipo. Advanced transfection with lipofectamine 2000 reagent. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338 100. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Your search returned transfection mdck cell line kits and reagents transfection reagents across 3 suppliers. Transient transfection of polarized epithelial monolayers with cftr. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different. Lipofectamine 2000 right panel around 5% efficiency. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Examples of cells transfected successfully thermo fisher. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. To perform transfection experiments in other cell culture plates, simply multiply the. While it delivers efficiently to many breast cancer cell types, it also maintains low cellular toxicity resulting in reduced experimental biases.
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. Improved delivery of cas9 proteingrna complexes using. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a comparable transfection efficiency when transfecting larger dna species scott et. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Invitrogen lipofectamine 2000 transfection reagent 15ml.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Cellspecific transfection protocols thermo fisher scientific us. The delivery of dna into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. For additional information on scaling your transfection reaction, see page 4. I want to try to use hela cell and lipofectamine 2000 to do transfection. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. L lipofectamine 2000 and incubated together with the cells in optimem i medium invitrogen for 4 h. Anyone familiar with transfection of mdck epithelial cells using paxillin, vinculin, zo1 and ecadherin plasmids.
I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Exceptional transfection efficiency in the broadest. Lipofectamine 2000 transfection protocol 10cm plates reagents. Genscript recommends using lipofectamine 2000 for all transfections. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. Highest transfection efficiency in cells at 90% confluency. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells.
Highest transfection efficiency in many cell types and formats. Plate cells at least 12hrs but not over 24 hours prior to transfection to achieve. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. A sample protocol is listed here for transfection experiments performed in 6well plates. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. A549 transfection kit lung carcinoma, ccl185 altogen. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. 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. Evidently, upon loss of cellcell contact between mdck cells, lipoplex binding. Dna complexes were added to cells that had already been allowed to. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Be sure to mix reagent invert 5 to 10 times prior to taking an aliquot for transfection.
I want to try to use hela cell and lipofectamine 2000 to. Transient plasmid dna transfection into hela and hacat cells. Anyone familiar with transfection of mdck epithelial cells using. General procedure for transfection of mammalian cells with. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine transfection of c2c12 cells bridges lab. Adhesion receptors mediate efficient nonviral gene delivery. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000.
Add 6l of lipofectamine2000 from biostores and 100 l of. Sep 01, 2009 the transfection procedure was as follows. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Conversion of mdck cell line to suspension culture by. Lipofectamine 3000 reagent thermo fisher scientific fr. Does lipofectamine 2000 give a stable transfection. Invitrogen lipofectamine transfection reagent protocols have been optimized. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. Lipofectamine 2000 transfection reagent from invitrogen.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Figure 1 illustrates a typical, reagentbased transfection protocol. This protocol is for transfecting plasmid dna into mammalian cells. Highest transfection efficiency in many cell types and formats e. 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. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transient plasmid dna transfection into hela and hacat. Invitrogen lipofectamine 2000 transfection reagent. Conversion of mdck cell line to suspension culture by transfecting. Hmec transfection kit mammary epithelial cells altogen. What percentage confluency should be used for stable transfection. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
Optimized conditions for successful transfection of human. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs. With cell number and dna concentration held constant, vary the amount of lipofectamine 2000 to determine the optimal concentration usually 1. Transfection protocol lipofectamine 3000 transfection. Do any one has protocol for stable transfection with lipofectamine 2000. I want to try to use hela cell and lipofectamine 2000 to do. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. The highest transfection efficiency in many cell types and formats e. Invitrogen lipofectamine 2000 transfection reagent fisher.
Lipofectamine 2000 and lipofectamine rnaimax transfection. Invitrogen lipofectamine 3000 transfection reagent l3000008. Numerous chemical and physical approaches have been used to deliver the dna, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Mdck, madindarby canine kidney, sirna, lipofectamine rnaimax, 778075. How does transfection with lipofectamine 2000 work. I am about to start my stable transfections with lipofectamine 2000. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. 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. A comparison showing transfection efficiency of polyjet reagent vs. Feb 18, 2016 to identify the best transfection reagents for delivery of cas9 rnps, initially we compared several commercially available protein transfection reagents, including lipofectamine 2000, lipofectamine 3000, lipofectamine rnaimax, lipofectamine meassengermax, turbofect, and xfect protein transfection reagent. Anyone familiar with transfection of mdck epithelial cells.
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