Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. For additional information, and protocols on transfecting suspension cells refer to the manual at. We are currently having technical issues with our pdf downloads on this page, we are very sorry for the inconvenience. Transfection protocol 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 invitrogen lipofectamine 3000 transfection reagent. Why is only lipofectamine the preferred way to introduce. If performing step 5, use dmem containing 10% fetal calf serum fcs for hek293 cells. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine 2000 and lipofectamine rnaimax transfection. Lipofectamine 2000 reagent thermo fisher scientific. For optimal transfection conditions with the mission sirna transfection reagent, the for 1.
Stealthsirna transfection protocol lipofectamine 2000. This reference provides general guidelines and procedures to transfect sirna. Can anybody give me exact protocol for sirna transfection. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Lipofectamine 2000 dna transfection reagent protocol. Can anybody give me exact protocol for sirna transfection using rnaimax. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and wait for 5 min at room temperature. Advanced transfection with lipofectamine 2000 reagent. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 2000 was simple to use and unlike other reagents displayed very low toxicity. Transfection protocol sirna resuspension guidelines we recommend preparing 10. Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein.
Rnai in cultured mammalian cells using synthetic sirnas. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. This protocol is well adjusted for cancer cell lines. Rnai transfection protocols thermo fisher scientific uk. How does transfection with lipofectamine 2000 work. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Rnai in cultured mammalian cells using synthetic sirnas ncbi. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines.
The protocol supplied by invitrogen was straight forward and also very accurate as we found that deviations from the protocol often resulted in lower transfection efficiencies and higher cell death. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Has anyone tried sirna transfection with lipofectamine 3000. Actinomycin d 1gml was added to hepg2 cells after 18hr transfection with either. Resuspend the sirna in nucleasefree water at a final concentration of 10. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
In contrast, 15 gfp did not efficiently transfect sirna into hela cells fig. Lipofectamine 2000 transfection reagent from invitrogen. Methods volume 33, issue 2, june 2004, pages 95103. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Why is only lipofectamine the preferred way to introduce sirnas. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell. The reagent provides high transfection efficiency in many cells types and formats. Transfecting sirna into mammalian cells using lipofectaminez 2000 introduction lipofectaminez 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Mission sirna transfection reagent s1452 technical. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 l volume. Reverse transfection of stealth rnai or sirna using lipofectamine 2000 in a 96 well plate format.
Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Transfection efficiency in a cancer cell line panel. In this video, we will perform an sirna transfection experiment using. L transfection of sirna to transfect cells with sirna, follow the protocol as described for dna but do not add p3000 reagent when diluting the sirna step 3. Can anyone recommend a protocol for transfection of hepg2. Lipofectamine 2000 has a broad range in nucleic acid. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. Add 1 ug of expression plasmid with 25 ul corresponding chimerarna 2 um solution in. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Has anyone tried sirna transfection with lipofectamine3000. Invitrogen lipofectamine 2000 transfection reagent.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics. Looking to do some preliminary experiments and i dont want to but a new reagent if i dont have to. Mammalian cell penetration, sirna transfection, and dna. The product manual includes advice on how to optimize transfection efficiency, as a number of conditions can be varied including dna. Briefly centrifuge the plate to ensure that the dried sirna is at the bottom of the wells. The complete synthesis protocols and experimental details can be found in the supporting information. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. I use 24well plates for transfections in triplicates. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. This protocol is for overexpression and knockdown at the same time. Transfection protocol lipofectamine 3000 transfection.
Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Lipidmediated dna and sirna transfection efficiency. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. This protocol is optimized for transfection of rko, an adherent cell line that. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Cells will adhere as usual in the presence of complexes. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection reagent life technologies, cat. Transfection and genome engineering fisher scientific.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Can anyone recommend a protocol for transfection of hepg2 cells. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. For many adherent cell types, we have observed that lipofectamine rnai max from thermo gives close to optimal results and recommend it for starting transfection optimization. See more supporting information at this feature is not available right now.
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