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 3000 transfection reagent kit youtube. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i. 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. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 3000 transfection benchling igem 2019. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Lipofectamine 2000 transfection reagent from invitrogen.
Transfection protocol lipofectamine 3000 transfection reagent. Do not add antibiotics to media during transfection as this will cause cell death. The protocol for lipofectamine 3000 lv production requires high cell density, 9599% confluence at the time of transfection. Thee two most important factors in choosing transfecting reagent are. Which round of collection may give better yield is it.
Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells important guidelines. Shop online for a wide selection of invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid fisher scientific. I use 24well plates for transfections in triplicates. To transfect cells with sirna, follow the protocol as described for dna but do not add p3000 reagent when diluting the sirna step 3. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection. Each reagent was used to transfect hela cells in a 96well format, and gfp expression was analyzed 24 hours posttransfection. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays.
This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Cas9 nuclease may be used in vivo to create targeted genome modifications. For additional information on scaling your transfection reaction, see page 4. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Has anyone tried sirna transfection with lipofectamine 3000. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 3000 transfection reagent lipofectamine 3000 reagent leverages our most advanced. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. We also found that hematopoietic cells were hard to transfect using lipofectamine 3000, lipofectamine rnaimax, and lipofectamine crisprmax. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. 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 3000 transfection reagent protocol transfect cells according to the following table. 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. Take care to maintain a standard seeding protocol between experiments because transfection efficiency is sensitive to culture confluence. A western blot was performed to determine the level of expression using actin as a control. With lipofectamine 3000, do i need to remove lipofectamine. 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. Transfection optimization and induction protocols for. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. To transfect cells with sirna, follow the protocol as.
Wholefield 60x view of hep g2 cells transfected with gwiz gfp plasmid dna and geneporter 3000 the intense gfp expression, combined with the 3x increase in. Make dnalipofectamine 3000 complexes in serumfree medium such as. Yes, you can avoid the polydlysine coating step when using lipofectamine 3000 as your transfection reagent to produce lv lentivirus. Original article an improved method for increasing the efficiency of. Transfection protocol lipofectamine 3000 transfection. Lipofectamine 2000 reagent thermo fisher scientific.
We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The protocal is from thermo fisher scientific important guidelines for transfection 1. With lipofectamine 3000, do i need to remove lipofectamine containing media after 6hours. This step generates a precipitate that is dispersed onto the cultured cells. Plate cells so they will be 7090% confluent at the time of transfection. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Aug 15, 2015 can i avoid coating step when i use lipofectamine 3000 to do the lv production. Invitrogen lipofectamine 3000 transfection reagent 1. Invitrogen lipofectamine 3000 transfection reagent 0. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Which round of collection may give better yield is it a problem, if i keep them overnight and replace. Discover the versatile reagent with the power to transfect your most difficult cells for just pennies per reaction the invitrogen lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. A highly efficient method of transfecting hep g2 cells summary. Improve lentiviral production using lipofectamine 3000. Use the indicated volume of dna and p3000 reagent with each of the 2 volumes of lipofectamine 3000. Lipofectamine 3000 reagent protocol thermo fisher scientific.
Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Lipofectamine3000 transfection of adherent mammalian cells. Lipofectamine 3000 protocol written out with timing specifics. A highly efficient method of transfecting hep g2 cells.
Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Transfection of cas9 rnp ribonucleoprotein into adherent. Use the indicated volume of dna and p3000 reagent with each of the two volumes of lipofectamine 3000 when. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. There are several ways in which to introduce cas9guide rna complexes into cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfectamine 5000 transfection reagent provided higher gfp transfection efficiency compared to lipofectamine 2000 and lipofectamine 3000 reagents.
Can i avoid coating step when i use lipofectamine 3000 to do the lv production. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Has anyone tried sirna transfection with lipofectamine3000. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Pool dishes of cells that have been prepared for transfection. According to our results, lipofectamine 3000, fugene and rnaimax. This protocol isintended for the transfection of adherent mammalian tissue culture cells. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The precipitate is taken up by the cells via endocytosis or phagocytosis. The protocol involves mixing dna with calcium chloride, adding this in a controlled manner to a buffered saline phosphate solution, and allowing the mixture to incubate at room temperature.
Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Even though cell lines differ in their experimental properties, this protocol will yield efficient transfection at low nano molar concentrations in most of the commonly. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Lipofectamine 3000 reagent thermo fisher scientific us. Add 6l of lipofectamine2000 from biostores and 100 l of. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Hepg2 cells were transfected with a vector expressing gsttagged stat protein. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and gfp expression was analyzed 48 hours posttransfection. Dec 31, 20 lipofectamine 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines. L3000001 lipofectamine 3000 transfection reagent sparshbio. Improve gene editing outcomes lipofectamine 3000 reagent was developed to break through. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000.
For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Contents transfection selection guide 4 transfection decision tree 5 superior transfection reagents 6. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat.
Imlf can be transfected using lipofectamine 3000 thermo fisher scientific, waltham, ma, usa but transfection efficiency is higher using recombinant adenovirus described later, which gives very good expression in a shorttime relative to other methods. Lipofectamine 2000 procedure for simultaneously transfecting. Lipofectamine 3000 protocol written out with timing. Lipofectamine 3000 reagent protocol lipofectamine 3000. Gibco hams f12 nutrient mix 11765054 5% horse serum. Feb 18, 2016 we also found that hematopoietic cells were hard to transfect using lipofectamine 3000, lipofectamine rnaimax, and lipofectamine crisprmax. Transfection and genome engineering fisher scientific. For each hardtotransfect cell line, we tested the delivery of cas9 rnps using the neon 24well optimization protocol supplementary table 4.
Improved delivery of cas9 proteingrna complexes using. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Lipofectamine 3000 transfection reagent hepg2 liver. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.