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. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. C first makes contact with endosomes, where tlr3 is localized, and only subsequently is it released in the cytosol where it interacts with cytosolic receptors. Oligofectaminez the purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or. Transfecting sirna into hela cells using oligofectaminez, continued limited use label license no. Versatile use of transfection reagents for short double.
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. Invitrogen lipofectamine 2000 transfection reagent life. Highly branched hk peptides are effective carriers of sirna. Lipofectamine 2000 and lipofectamine rnaimax transfection. Fast and efficient transfection of mouse embryonic stem. Prior to joining life technologies, henry worked in the fields of gene therapy and viral genetics. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and oct4, a. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Thermo scientific dharmafect transfection reagents deliver specialized formulations for successful delivery of sirna to more cell lines.
Transient and stable vector transfections have played important roles in illustrating the function of specific genesproteins. Lipofectamine 2000 transfection reagent from invitrogen. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Whats the difference between lipofectine and lipofectamine 2000 reagents. Tas for 6 hr, a according to the companys manual or b and e with slightly changed. Nov 28, 2016 we set a single linear region as shown in fig.
The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Different sirna transfection agents have enyone tested. 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. Oligofectamine reagent 1 is a proprietary formulation that is suitable for the transfection of oligonucleotides into eukaryotic cells. Rapid and highly efficient mammalian cell engineering via. In contrast, carriers of sirna such as oligofectamine and lipofectamine 2000 were significantly more toxic. Pdf highly branched hk peptides are effective carriers. Invitrogen lipofectamine 2000 transfection reagent. Prewarm 50ml of optimum stored in cold room at 4c 10min. Oligofectamine to substitute lipofectamine in crispr. Transfecting sirna into hela cells using oligofectaminez. The intracellular trafficking mechanism of lipofectamine.
Evaluation of transfection methods for transient gene. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Whats the difference between lipofectine and lipofectamine. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Liposome lipidbased formulation has the least influence on raav. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. Lentiviral production protocol overview lipofectamine messengermax reagent is an mrna transfection reagent that offers superior transfection ef. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Plasmid dna and synthetic rnai into mammalian cells using lipofectamine 2000. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. 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. Transittko, oligofectamine reagent, or lipofectamine 2000 diluted in serum free dmem. Ive been using oligofectamine for sirna transfection in adherent cells. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
Do you have experience using an efficient transfection. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Formulation of lipofectamine 2000 and lipofectin signagen blog. 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. Oligofectamine transfection reagent forms stable complexes with oligos, permitting efficient transfection into eukaryotic cells in a highly specific, yet nontoxic fashion. An efficient sirna transfection reagent in human embryonic stem cells, molecular biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Downregulation enhanced green fluorescence protein gene. Oligofectamine transfection reagent is a proprietary formulation for transfecting oligonucleotides and short interfering rna sirna into eukaryotic cells.
Our previous studies have shown that the lipofectamine rnaimax is a more efficient transfection reagent for delivering sirna into hes cells compared with oligofectamine and lipofectamine 2000 31. Transfection efficiency in a cancer cell line panel. Thermo scientific dharmafect transfection reagents. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added.
Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Scaling up or down lipofectamine 3000 reagent transfections. Dnaplus lipid complexes must be made in serumfree medium such. Smallsized, stable lipid nanoparticle for the efficient. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. If you are studying rnai oligos, the following link have different transfection protocol for different cell types. The general assumption is that such a platform could effectively link a given geneprotein to gained phenotypes, revealing the mechanism of how a gene works. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Not recommended for use with lipofectamine 2000 or lipofectamine rnaimax. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Hepatocellular carcinoma sixth most common cancer third leading cause of cancer related deaths early stage treatments liver transplantation and surgical resection metastasis and recurrence dna will be transfected into the huh7 cell line ability to grow without serum growth. Nucleic acid lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. I use lipofectamine 2000 as my standard transfection reagent of choice and found that it. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and.
Mirus lipofectamine 2000 and oligofectamine invitrogen. I use 24well plates for transfections in triplicates. Antibiotics during sirna transfection sirna, microrna. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Liposome lipidbased formulation has the least influence. The results demonstrated that egfp expression was significantly and. Comparison of transfection through lipofectamine and polyeth. It was known that it is harder to transfect plasmid into endothelial cells. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Lipofectamine 3000 reagent thermo fisher scientific kr. The single linear regions were set under conditions that the value of %gated in the histogram of nt cells was equal to or less than 1%. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Do you have experience using an efficient transfection reagent for.
For additional information on scaling your transfection reaction, see page 4. Do you have experience using an efficient transfection reagent for mimics mirna better than lipofectamine. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. We are using oligofectamine from invitrogen with great success. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Most publications are using lipofectamine 2000 and oligofectamine invitrogen for rnai studies. Cell type, source, lipofectamine 2000, lipofectamine rnaimax, oligofectamine. Therefore, transfection rates and success between cell types may be more. He received his phd from harvard university and did his postdoctoral training at the university of. Lipids are a broad class of fatsoluble biomolecules, such as fats, oils, and waxes. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Can anybody give me exact protocol for sirna transfection using. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Invitrogen has the most complete collection of transfection reagents with. Invitrogen lipofectamine 2000 transfection reagent fisher. The commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. The protocal is from thermo fisher scientific important guidelines for transfection 1. Different sirna transfection agents have enyone tested them. Lipofectamine 2000 invitrogen was used in plasmid transfection and plasmid plus rna co tranfection. Oligofectamine transfection reagent thermo fisher scientific. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and oct4, a key regulator of pluripotency. Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Highest transfection efficiency in many cell types and formats e.
May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Invitrogen lipofectamine 2000 transfection reagent 1. Remarkably, significant changes in gene expression was also observed after treating the cells with pdna lipofectamine 2000 complex for 8 hour while no change in cellular. Im about to transfect viral rna from an in vitro transcription assay by cationic liposomes, into bhk cells and i was. Transfection and genome engineering fisher scientific. Lipofectamine 2000 reagent thermo fisher scientific.
Rnai max reagent from life technologies works awesome just follow their manual. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000, 3000, messengermax, and rnaimax. Add 6l of lipofectamine2000 from biostores and 100 l of. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. The trusted, simple, broadspectrum transfection reagent for most cell lines. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transittko, oligofectamine reagent, or lipofectamine 2000 diluted in serumfree dmem. 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. C is complexed with lipofectamine, its delivery into the cell is not directly to the cytosol, but, once internalized, polyi. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
Im seeing a lot of cell death with my current protocol and think it might be because of the. 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. With only a fiveminute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Has anyone tried using oligofectamine to transfect cells in crispr, rather than lipofectamine. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Stealthsirna transfection protocol lipofectamine 2000. 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.
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