293 transfection protocol lipofectamine 2000 manual

To transfect cells with sirna, follow the protocol as described for dna but. The hek293t cell line was generated more than 30 years ago, which has. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and. Tm409 viafect transfection reagent technical manual. General procedure for transfection of mammalian cells with. For consistent animal originfree transfection, use optipro sfm cat.

If you can see successful transfection through a visual diagnostic e. Transfection protocol lipofectamine 3000 transfection. Kit qiagen, hilden, germany according manufacturers instructions and the dna. The transfection reagent, lipofectamine2000, was selected for. Transient plasmid dna transfection into hela and hacat cells. Cd 293, 293 sfm ii, cd hybridoma may inhibit cationic lipidmediated transfection. Hek 293 cells are efficiently transfected with lipofectamine. Rnai in cultured mammalian cells using synthetic sirnas ncbi. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 2000 reagent thermo fisher scientific. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent. Hek293 standard transfection protocol 24well plate.

Maintain p19 cells undifferentiated in mem with 10% serum 7. Expi293 expression system thermo fisher scientific. Atcc achieved transfection efficiencies of 99%, using the protocol described below. Lipofectamine 2000 dna transfection reagent protocol. Expifectamine 293 transfection kit protocol see page 7 to view a typical transfection procedure. Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein compared to the. Tm328 fugene hd transfection reagent technical manual. Transfection conditions for expi293f cells for each 30ml transfection, use 7.

Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Ian chapman shows us how to transfect mammalian cells using fugene in a 12well plate. To transfect cells with sirna, follow the protocol as. According to the manufacturer, and my own experience using human embryonic kidney cells, hek 293, lipofectamine 2000 fulfills both of these criteria. If you are using larger or smallersized tissue culture plates, vary the transfection conditions i. Scale your transfections up or down by proportionately adjusting the.

Using the transfection reagent lipofectamine 2000, two different. Lipofectamine 2000 transfection protocol 10cm plates reagents. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules. Lipofectamine 2000 transfection reagent from invitrogen. The protocol for a 24well transfection reaction with hek293 cells is here. A guide to transient expression of membrane proteins in. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection protocol transfection components component cat. Freestyle 293 expression system for largescale transfection of suspension 293 cells in a defined, serumfree medium freestyle max reagent human mesenchymal stem cells lipofectamine 2000 lipofectamine 2000 lipofectamine reagent for the transfection of dna into eukaryotic cells.

Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Can anybody give me exact protocol for sirna transfection using rnaimax. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. You can not use antibiotics during transfection step because it will cause cell death. The prepared mix is enough to have triplicates 96well, duplicates 24well, and single well 6well transfections, and account for pipetting variations. Transient plasmid dna transfection into hela and hacat. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 3000 protocol written out with timing. The protocol for use of expifectamine 293 transfection components is very similar to transfection protocols for other transient expression reagents, such as 293fectin and freestyle max.

Transient transfection of mammalian cells with lipofectamine. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Freestyle 293 expression system for largescale transfection of suspension 293 cells in a. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol. Protocols for transfecting the most common cell lines with xtremegene transfection reagents. Transfection guide overview of transfection methods. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production. Transfection protocols thermo fisher scientific in.

Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. According to the manufacture manual, pcr products were first mixed with 10. Use the following protocol to transfect adherent 293 cells in 24well plates. Transfection and genome engineering fisher scientific. Gfp expression was analyzed 48 hours posttransfection. Can anyone provide me with a proper transfection protocol into. A guide to transient expression of membrane proteins in hek 293 cells for functional characterization. The enhancers are added 16 to 18 hours post transfection to boost transfection, cell viability, and protein expression. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Plate cells so they will be 6080% confluent at the time of transfection. Transfection efficiencies lower than 50% were not reported in this table. Protocols for transfecting the most common cell lines with. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.

I am doing transfection using lipofectamine 2000 and plasmid dna, too. General considerations for using the transfex transfection reagent. Tell me about your experiences in transfection please, thanks. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Transfex transfection of plasmid dna into hek293t17 cells. Plate cells so they will be 7090% confluent at the time of transfection. For additional information on scaling your transfection reaction, see page 4. Please be aware that the protocols are suggestions and that optimal conditions must be determined empirically. For example, one reagent may work well with hek293 cells, but another. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Can anyone provide me with a proper transfection protocol. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.

Each reagent was used to transfect hek 293, hela, lncap, a549, and. I do have cell toxicity, but not so serious as yours. Original article an improved method for increasing the efficiency of. Hek293 cells exhibited the highest egfp expression compared to the bj.

Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 transfection reagent life technologies, cat. I have ordered lipofectamine 2000 from thermo fishers scientific. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. How can i increase shrna transfection efficiency in. Optimized conditions for successful transfection of human. 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. How can i increase shrna transfection efficiency in hek293t cells. Higher densities can lead to spontaneous differentiation. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. I use 24well plates for transfections in triplicates.

Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. We use it regularly for a range of cell lines such as hek 293, cho and cos7 and obtain consistently greater than 90% efficiency. Expi293 expression system user guide for scalable transfection of expi293f cell lines in a chemically defined, serumfree medium, using the expifectamine 293 transfection kit for scalable metabolic protein labeling using expi293f cell lines, in a chemically defined, serumfree, methioninefree medium using a. Using hek293t expression system to study photoactive. However, i could only reach at most 2030% transfection efficiency. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The standard transfection protocol while resulting in better transfection. Transfection guide overview of transfection methods promega. The product has been provided with a protocol, but does not clearly reports. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane.