The human embryonic kidney 293 hek293 cells are commonly. I have been trying to transfect hek293 cells with transfast and lipofectamine and have not been able to confirm successful transfection. Plate cells so they will be 7090% confluent at the time of transfection. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Scaling up or down lipofectamine 3000 reagent transfections. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Volume glutamax gibco 35050 5 ml penicillinstreptomycin gibco 15140122 5 ml fbs gibco 26140079 50 ml 10%. Using hek293t expression system to study photoactive.
It is used to increase the transfection efficiency of rna. Lipofectamine 2000 is that it has an extensive, very useful manual. 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. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Cotransfection of two plasmids tissue and cell culture. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1, which is in agreement with the recent finding that rnaimax performed better than lipofectamine 2000 for.
Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectaminez 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium. Lipofectamine2000 works quite well in 293cells 90%. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Transfection of hek and bj cells with lipofectamine 2000. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells.
The lenti293t cell line has been tested as a suitable cell line for lentiviral production. Carefully transfer the transfection mix to the lentix 293t packaging cells. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. I am using lipofectamine 2000 with optimem to transfect hek cells, but i havent had much luck so far. Advanced transfection with lipofectamine 2000 reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Rapid and highly efficient mammalian cell engineering via. 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. Transfection efficiencies lower than 50% were not reported in this table. Nfat response element stably integrated into hek 293t cells. According to the manufacture manual, pcr products were first mixed with 10. Incubate the cells for 18 h, or until the following. Cloned genes can be transfected into cells for biochemical characterization. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells complete growth medium component cat. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 3000 has a reduced cytotoxicity in comparison to lipofectamine 2000. For additional information on scaling your transfection reaction, see page 4. 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.
Transfection protocol of lipofectamine rnaimax can be. Dna complexes were added to cells that had already been allowed to. Transfection protocol lipofectamine 3000 transfection. Maintain p19 cells undifferentiated in mem with 10% serum 7. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Strong expression of egfp was observed in hek293 cells, mouse. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Original article an improved method for increasing the efficiency of. Transfer the transfection mix 500ul to 293t cells in seeding. Rnai or technical service, as are celltype specific rnai transfection protocols see rnai protocols. Optional the final cell density for transfection protocol varies.
I tried both of them and lipofectamine 2000 was much betterin 293ft cells. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Invitrogen lipofectamine 2000 transfection reagent. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. I have ordered lipofectamine 2000 from thermo fishers. Add the transfection mix dropwise being careful not to dislodge the cells. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Can anyone provide me with a proper transfection protocol into. Lipofectamine 3000 reagent thermo fisher scientific us. Invitrogen lipofectamine 2000 transfection reagent 15ml. Comparative transfection efficiency of jetprime versus its main competitor.
Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format, using 0. Higher densities can lead to spontaneous differentiation. I would like to transfect 293t cells with lipofectamine 3000. Lipofectamine 2000 transfection reagent from invitrogen. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. For transfection, i mix 1 ug of dna in total plus 2. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. I normally incubate my cells in media with antibiotics and serum, and a.
How to transfect hek293 cells using lipofectamine 3000. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. We produce lenticdna viral stocks in 293t cells using the transfection conditions. Optimized conditions for successful transfection of human. The multiple cloning sites mcs sequence in plv cdna vectors. Add 6l of lipofectamine2000 from biostores and 100 l of. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different.
Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 transfection protocol 10cm plates reagents. Transfection of 293 cells in 10 cm dishes abbreviated protocol surface area of 15 cm dish is 18 x greater than that of a 6well dish. Prewarm 50ml of optimum stored in cold room at 4c 10min.
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