I use 24well plates for transfections in triplicates. Im about to transfect viral rna from an in vitro transcription assay by cationic liposomes, into bhk cells and i was. Lipofectamine 2000 and lipofectamine rnaimax transfection. Enhanced transfection of cryomanipulated lf2000 is not dependent on. Highest transfection efficiency in many cell types and formats. Transfection with lipofectamin 2000 invitrogen culture vessel 96 well 48well 24well 12well 6well 35 mm 60 mm 100 mm 150 mm t25 t75 surface area cm2 0. Lipofectamine 2000 transfection reagent thermo fisher scientific. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy.
After 24 h of transfection, invitrogen trizol reagent thermo fisher. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Rnaimax transfection reagent 778030, invitrogen, waltham, ma. Primary cortical cultures on div78 or neuroblastoma cells b35 and b104 were transiently transfected using lipofectamine 2000 invitrogen.
The trusted, simple, broadspectrum transfection reagent for most cell lines. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Cells were harvested with trizol reagent invitrogen. Lipofectamine 2000 from invitrogen biocompare product. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 2000 dna transfection reagent protocol. Dnaplus lipid complexes must be made in serumfree medium such. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format. Shop online for a wide selection of invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3.
Optimizing transfection lipofectamine 2000 cd reagent. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. The multiple cloning sites mcs sequence in plv cdna vectors. Lipofectamine 2000 transfection reagent this product. 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 and suspension cell lines. Neon device invitrogen lipofectamine 2000 invitrogen unlike the other devices, the nepa21 requires no buffers. 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. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Lipofectamine 2000 transfection reagent is a proprietary formulation. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. A kozak consensus sequence gccacc before start codon is highly recommended. 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. The advantages of lipidbased transfection reagents have permitted. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Evaluation of transfection methods for transient gene. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. The intracellular trafficking mechanism of lipofectamine. Invitrogens lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Lipofectamine 2000 transfection protocol 10cm plates reagents. Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 reagent thermo fisher scientific.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. The average lentiviral titers in our preparations are around 5 x 10. The transfection reagent lipofectamine 2000 lf2000, invitrogen is. Whats the difference between lipofectine and lipofectamine 2000 reagents. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 is commonly used for sirna transfections. Invitrogen lipofectamine 2000 transfection reagent. On measuring mirnas after transient transfection of mimics. The epub format uses ebook readers, which have several ease of reading. The transfection efficacy of lipofectamine 2000 was compromised by.
Contact life technologies technical service or your local sales representative for permission to crossreference the dmf. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000pgfpn2 transfection. Pdf a wide variety of mammalian cell types is used in gene transfection studies. Invitrogen lipofectamine 2000 transfection reagent 1. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Effects of lipofectamine 2000sirna complexes on autophagy. Estimating transfection efficiency in differentiated and. Pdf an electroporation protocol for efficient dna transfection in. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. To this aim, we used complexes of lfn 2000 with cy3labeled.
Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. All cell lines were transfected using lipofectamine 2000 invitrogen in 24well. The ratios of sirna to lipofectamine 2000 used were 0. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. Can anyone recommend a protocol for transfection of hepg2 cells. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Effects of tlr4 gene silencing on the proliferation and apotosis of. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Product isguaranteed for 6 months from the date of shipment if stored properly. However, few studies have examined cellular responses to this delivery system. Comparison of nepa21 with amaxanucleofector, neon and lipofectamine 2000. Comparison of hsv, adenovirus, aav, retrovirus and lentivirus for gene delivery.
Life technologies corporation andor its affiliates warrant their products as set. The intracellular trafficking mechanism of lipofectaminebased. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Request pdf advanced transfection with lipofectamine 2000 reagent. 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. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. Photograph of expected results dna lipofectamine scaling up or down. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Optimized conditions for successful transfection of human. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon. Try our upgraded product lipofectamine 3000 reagent. Lipidbased transfection reagents exhibit cryoinduced increase in.