Lipofectamine 3000 reagent outperforms lipofectamine 2000 and. Overview this protocol is for transfecting plasmid dna into mammalian cells. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the. Using hek293t expression system to study photoactive. 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 used cell lines, such as hela, a549, mcf7 or hek293. This is often the best place to start, especially in a new cell line. Optimization of transfection methods for huh7 and vero. Hek293t human embryonic kidney cell line 293t crl3216, from the atcc cell bank, usa or 293t acc 635 from the dsmz cell bank, germany. Also prepare the sterile dna for transfection 2030 g10cm dish70 for a 15cm dish.
The dna lipofectamine 2000 complexes were then added to each well containing cells and medium. Hek293 cells were cultured in dmem media and transfected with ptgs2. To optimize transfection of your stem cells in your culture conditions, we recommend starting with two amounts of dna and two amounts of lipofectamine stem transfection reagent following the chart below. 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. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Optimized conditions for successful transfection of human. Gene transfection and western blotting protocol day 1 transfection calciumphosphatemediated transfection v prepare solution a and b solution a. Volumes are listed on a per well basis in a 24well format. Transfection of human hek 293f cells with metafectene.
Transfection protocol lipofectamine 3000 transfection. Although several transfection protocols have been successfully used for the production of lvv 8910, most of them involve expensive transfection reagents e. The xfect rna transfection reagent can be used to enhance trasfection with single guide rna sgrna for crisprcas9mediated gene. 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. I am currently having problems with my transfections and request help. Prior to transfection, hek 293 cells are preincubated with serumfree dmem medium for 1 h. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Prepare the transfection buffer as noted in the material section. On the day of transfection, 500 ng cas9 protein and 120 ng hprt1 grna were transfected with either lipofectamine 2000 lf2k, lipofectamine 3000 lf3k, lipofectamine messengermax, lipofectamine rnaimax, turbofect or xfect transfection reagent.
Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Pdf polyethyleniminebased transfection method as a. A specific, precalculated amount of the reagent needs to be complexed with dnarna in optimem medium. Dna ratios and when using easytotransfect cell lines such as hek 293 cells, dnain and lipofectamine 2000 achieved similar transfection efficiencies i. Lipofectamine 3000efficient, reproducible transfection for. Thrombin and lysophosphatidic acid receptors utilize distinct. Generating mutant renal cell lines using crispr technologies.
Pdf transfection optimisation in hek293 cell line researchgate. Can anyone provide me with a proper transfection protocol. Protocol 2 producing lentivirus in hek293t cells using a. Figure 22comparison of the transfection efficacy of pmaxgfp with chemical reagents effectene, lipofectamine 2000, lipofectamine plus, and. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Western blot showed the strongest egfp expression using cell lysates from lipofectamine 3000 transfected hek293 cells and transduced huvecs compared with lipofectamine 2000 or fugene 6 reagents. Feb 08, 2010 with the exception of hek 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering dna into the cells as determined by cell proliferation and gfp expression, respectively. Plate cells so they will be 7090% confluent at the time of transfection. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate.
The protocol can be scaled to produce different amounts of virus as needed. Trojanporter was used as described in the protocol for a 6well plate. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine 2000 dna transfection reagent protocol. If you find this doesnt work for your specific cell type, then you can look to our cellspecific protocols below for further optimization. Choosing the right transfection reagent for optimal efficiency. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Would anyone have any efficient protocol for transfection of the pcdna3.
Pdf polyethyleniminebased transfection method as a simple. The examples given within the protocol are for 96well plates, and table 1 provides the transfection reagent volumes for additional plate types. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of. Hek293 cells, bj fibroblasts, and ecs were transfected with 1.
An improved method for increasing the efficiency of gene. I want to try to use hela cell and lipofectamine 2000 to do. The transfection efficacy of lipofectamine 2000 was compromised by. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. For each well, 4 g of circular retrovirus dna was added to l of. D after 20 min incubation a add the dna lipofectamine mix to the cells 1 mla 5 ml, the total medium will be 6 ml. Pdf comparative transfection of dna into primary and. This protocol isintended for the transfection of adherent mammalian tissue culture cells. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. In our experience, hek293t cells are easily transfected and the percentage of transfection can be around 80% by using the present protocol. Transfecting sirna using lipofectamine rnaimax pin liu, 2017. Hek293 and pc3 cells express all three of these pro teins with. They are also known, more informally, as hek cells.
Transfection of hek and bj cells with lipofectamine 2000. Lipofectamine 2000 transfection reagent from invitrogen. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. This is necessary because serum is replete with rnases that degrade. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Total ml 2000 add methanol after mixing 10x transfer buffer and h 2o, then fill to or 2000 ml using h 2o. An abbreviated protocol for transfecting editr synthetic guide rna and cas9 mrna into cultured mammalian cells using dharmafect duo transfection reagent view protocol knockin protocol. Optimization of chemically based transfection conditions in huh7 and vero cel ls and flow cytometry plots of the trans fected cells.
Invitrogen lipofectamine transfection reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Feb 18, 2016 identification of lipofectamine crisprmax. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection efficiency with different ratios of dna. C incubate at room temperature for 5min a combine the dna mixture with diluted lipofectamine 2000. Dharmafect transfection reagentssirna transfection. Herein, we report the transfection efficiency and toxicity. 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 used cell.
Lipofectamine 2000 from invitrogen biocompare product. Recommended transfection protocols for 24well plate. Hek293 standard transfection protocol 24well plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection efficiency and protein expression using geneart crispr nuclease vector. Lipofectamine rnaimax has a broad peak of activity. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Maintain p19 cells undifferentiated in mem with 10% serum. Transfection of human hek 293f cells with metafectene pro. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Seed cells onto the wellplates petridishes to be transfected so that day 1 is at 7080%. Optimization of transfection methods for huh7 and vero cells.
Resuspend at 2 gml, and take the od on a small sample a 1. Make sure that cells are greater than 90% confluent and vary dna. Hek293 is a cell line derived from human embryonic kidney cells grown in tissue culture. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Lipofectamine 2000 transfection reagent thermo fisher.
Would anyone have an efficient protocol for transfection. Improved delivery of cas9 proteingrna complexes using. Forward transfection use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see. Mar 27, 2007 according to the manufacturer, and my own experience using human embryonic kidney cells, hek 293, lipofectamine 2000 fulfills both of these criteria. For your convenience, the essential components of this protocol are now available in the gibco prostate cancer starter kit. Hek293 cell transfection 96wellplate format with lipofectamine 2000. They look nice for a couple of hours after transfection, when i change to full medium 6h.
Dharmafect volumes per well represent guidelines and need to be optimized. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Perform the transfection in the late afternoon because the transfection mix should only be incubated with the cells for 1215 hours. Lipofectamine 2000 d after 20 min incubation add the dnalipofectamine mix to the cells 1 ml 5 ml, the total medium will be 6 ml 4. In polypropylene microfuge tubes do not use polystyrene tubes, make a cocktail for each transfection.
Dharmafect transfection reagentssirna transfection protocol. Use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see scaling up or down transfections, page 3. Gene editing sgrna px330 imcd3 hek293 transfection fluorescence activated. Lipofectamine and nucleofection of hek 293 were essentially equivalent in terms of toxicity and efficiency.
Protocol 2 producing lentivirus in hek293t cells using a 2. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. For hek293, lipofectamine 3000, fugene and rnaimax showed the highest transfection. The key to obtaining a high transfection efficiency is twofold.
Each reagent was used to transfect hek 293, hela, lncap, a549, and. Dharmafect sirna transfection protocol horizon discovery. Hek293 cells are useful for many transfection experiments, particularly the propagation of adenoviralbased and retroviralbased vectors. Would anyone have an efficient protocol for transfection of. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. I use lipofectamine 3000 to transfect hek 293 cells in 6 wells plates. The mechanism of lipofectamine 2000 mediated transmembrane. Why do hek293t cells detach after transfection with. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection reagent, we can achieve about 90% cx43 knockdown efficacy in huvecs. Comparative transfection of dna into primary and transformed.
Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. We offer a range of xfect transfection reagents that are known for superb performance, low toxicity, and userfriendly protocols. The transfection reagent, lipofectamine 2000, was selected for. Plate cells so they will be 6080% confluent at the time of transfection. Each reagent was tested with a range of transfection reagent. Hek293 cell lines were maintained in dulbeccos modified.
Do not add antibiotics to media during transfection as this causes cell death. Hek 293 cells are popular for their ease of growth and transfection hek293 transfection kit, making them a common cell culture in cancer research. According to the manufacture manual, pcr products were first mixed with 10. In addition, high transfection efficiency of hek293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes.
Protocol the following is a general protocol for use of dharmafect transfection reagents to deliver sirna into cultured mammalian cells. Transfection and ancillary reagents horizon discovery. The transfection efficiency and toxicity is equivalent following nucleofection or lipofectamine transfection of hek293 cells figure 1. Transfection of two immortalized human airway epithelial cell lines, 16hbe14oand cfb41oand primary airway epithelial cells from pig and human pte and hte, respectively showed that nucleofection was more. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. Transfection protocol lipofectamine 3000 transfection reagent. The following is a general protocol for use of dharmafect transfection. Add 6l of lipofectamine2000 from biostores and 100 l of.
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