Cas9 protein and guide rna can both be encoded as plasmid dna for transfection. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Plate cells so they will be 6080% confluent at the time of transfection. Improved delivery of cas9 proteingrna complexes using. Viafecttm transfection reagent technical manual tm409. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Rnaimax reverse transfections lipofectamine thermo. Follow manufacturers handbook protocol unless otherwise indicated. This protocol is for a basic assay using an xcelligence rtca instrument to monitor the effects of sirna knockdown. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. How can i increase shrna transfection efficiency in. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Print bookmark share the next generation in lipid technology pdf 4kb english format file size language download get adobe reader.
Universal transfection reagent protocol sigmaaldrich. Using 384well plates has the advantage of significantly reducing. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Protocol for transfection of mes using lentivirus reply. The unique properties of the delicate cells make transfection challenging and necessitate specialized techniques. Invitrogen lipofectamine 2000 transfection reagent. It describes transfection of mammalian cells with rna mrna, sgrna, microrna, or shrna in a 12well plate format. Kenji yamato at tokyo medical and dental university protocol 1. If you find this doesnt work for your specific cell type, then you can you look to our cell specific protocols for further optimization.
For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. This is often the best place to start, especially in a new cell line. 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, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results.
Cookie information is stored in your browser and performs functions such as recognising you when you return to our website and helping our team to understand which. If you choose not to optimize transfection parameters, use the general conditions recommended below. As for all transfection methods, electroporation has its advantages and disadvantages. Designed to transfect a broad range of eukaryotic cells, including insect cells, many cell lines not transfected well by other reagents, and hardtotransfect cell lines. Sigmas universal transfection reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardtotransfect primary cells. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The protocol given here is modified from a previously published protocol used for solidphase transfection in a 96well format erfle et al. Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 7080% confluency at the time of transfection. Transfection protocol lipofectamine 3000 transfection reagent t84 lung cancer cells complete growth medium component cat. On the plus side, you can use it to transfect large dna fragments and achieve good transfection efficiencies using cell lines. The optimal transfection conditions for a majority of adherent cell lines, as well as a general starting point for optimization are given in the standard protocol described below. The next generation in lipid technology pdf 4kb english format. Transfection protocol transfection components component cat.
Transfection the transfer of genetic material into mammalian cells is a valuable tool that allows scientists to genetically manipulate neurons and neuronal tissue. I use 24well plates for transfections in triplicates. Successful transfection of each cell type requires optimization of the basic protocol. Lipofectamine transfection of c2c12 cells bridges lab. 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. Variables to consider for optimization include, but are not limited to, cell density, transfection reagent, duration of transfection, and dna concentration. With modern genetic techniques allowing scientists to identify the link between genes and diseases, there is an enormous emphasis on determining ways to alter the function of a gene in vivo. Use the following table to scale the volumes for your transfection experiment.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection protocol lipofectamine 3000 transfection. Transitlt1 transfection reagent is a broad spectrum reagent that provides high efficiency plasmid dna delivery in many mammalian cell types including. Viafect transfection reagenta is a novel formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low. For optimal transfection, we recommend testing rnp concentrations ranging from 10 to 30 nm final in well. Sep 10, 20 so, in our experiments using lipofectamine rnai max to support our rnai based screening we are very happy to be able to go through quite a number of cell lines, quite a number of gene targets in. Xtremegene hp dna transfection reagent sigmaaldrich. The following protocol is given for transfection in 24well plates, refer to table 2 for transfection in other culture formats. Deaedextran is a cationic polymer that tightly associates. As cells are inherently variable, some optimization of the protocol may be necessary to achieve maximum transfection efficiency. Plate cells so they will be 7090% confluent at the time of transfection. Comparison of small interfering rna sirna delivery into.
Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Chemical reagents one of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells was deaedextran vaheri and pagano, 1965. Mission sirna transfection reagent s1452 technical bulletin. Indeed, some mirna are also known to induce gene expression by association with the promoter of the gene of interest. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. I have tried transfection as per protocol, but it seems the cells had toxic effect on them post transfection. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Transfection protocol lipofectamine 3000 transfection reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro.
To simplify the transfection optimization process, we have created a transfection. The insertion of dna into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of rna into a cell is used to downregulate the production of a specific protein by stopping translation. This protocol ataglance is provided for transfection of cells with rna using the xfect rna transfection reagent cat. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. Lipofectamine 2000 dna transfection reagent protocol. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. It may also refer to other methods and cell types, although other terms are often preferred. Transfection protocols thermo fisher scientific in. Amaxa 4dnucleofector optimization protocol for cell lines. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels. Lipofectamine rnaimax offers a simple protocol no need to remove transfection complexes, or change or add medium following transfection.
Approximately 1824 hours before transfection, plate cells using the following guidelines. Lipofectamine 2000 and lipofectamine rnaimax transfection. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transient transfection of cos1 cells upstate this protocol has been optimized for cos1 cells. Transfection of drosophila melanogasters2 cells qiagen. On the day of transfection just prior to preparing complexes, plate 1. Lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Transfection protocol transfection procedure this procedure is designed for sirna in combination with lipofectamine rnaimax. Effectene reagent yields high drosophila melanogasters2 cells. However, all the transfection reagents tested induced an ifn response in the absence of sirna, which could be minimized by reducing the transfection reagent incubation period.
Scaling up or down lipofectamine 3000 reagent transfections. Though this protocol has been optimized using the a549 human nonsmall cell lung cancer cell line, assay conditions may require addi. Invitrogen lipofectamine transfection reagent protocols have been optimized. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. This website uses cookies so that we can provide you with the best user experience possible. For plate size volume of sirna transfection reagent per well l reactions 1. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine transfection of c2c12 cells bridges lab protocols. The prepared mix is sufficient for triplicate transfections with overage. Plate cells to obtain 7080% cell density on the day of transfection. Unfortunately, no single delivery method or transfection reagent can be applied to all types of cells. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection.
Reverse transfection using ntersirna nanoparticles. Reverse transfection an overview sciencedirect topics. Lipofectamine rnaimax transfection reagent from thermo fisher. The total volume of transfection complex medium, dna and viafect transfection reagent to add per well of a 96well plate is 510. The newer transfection reagents lipofectamine 2000 dalby et al. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Celltype specific transfection protocols are available at our website or through technical service. Our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Transfection protocol lipofectamine 3000 transfection reagent t84. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Silencer select sirna transfection with lipofectamine rnaimax.
The protocol type is indicated immediately under the title of the protocol. Cell confluency, reagent volume, and post transfection incubation time are a few key parameters that affect the outcome of transfection experiments. Lipofectamine rnaimax transfection reagent delivery of. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics.
Reverse transfection protocol acea biosciences inc. 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. Prepare the transfection buffer as noted in the material section. Oligofectamine reagent 1 is a proprietary formulation that is suitable for the transfection of oligonucleotides into eukaryotic cells. Same protocol as above except use a961 plasmid instead of a979 plasmid. Strategies to treat these diseases include delivering an alternate gene to the cell, replacing the defective gene or silencing the defective gene using rnai or. Protocols based on wetlab experiments have provided successful results when used by qiagen scientists for transfection of the cell type.
Furthermore, with only half the quantity of dna used for the calcium phosphate method i. Alternatively, cas9 can be delivered as plasmid dna, and guide rna can be supplied as an rna oligonucleotide. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. 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. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. For formats other than 12well plates, see tables i and ii for appropriate reaction volumes. Transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a wide range of cells invitrogen lipofectamine stem transfection reagent. Reverse transfection is the protocol of choice for most applications, as it frequently yields better transfection efficiency and facilitates the experimental protocol by combining cell seeding and transfection into a single step.
A preoptimized a549 transfection kit is available from altogen biosystems, which includes. Other cell types or other formats should be adjusted to achieve optimal cell density for transfections. Timeline steps procedure details day 0 1 seed cells to be 60. Amaxa 4dnucleofector optimization protocol for cell lines for 4dnucleofector x unittransfection in suspension for use with plasmid dna andor sirna note the cell line optimization protocol enables you to optimize 4dnucleofection conditions for a cell line of your choice using our cell line optimization 4dnucleofector x kit. Can anyone recommend a protocol for transfection of hepg2. Highest transfection efficiency in cells at 90% confluency. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l.
Promega corporaoon 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526. With its ability to transfect a broad range of cell types, lipofectamine rnaimax reagent, combined with the invitrogen blockit alexa fluor red fluorescent control, gives you the most versatile approach to all of your knockdown experiments table 1, figure 3. Cellspecific transfection protocols thermo fisher scientific us. To transfect cells with sirna, follow the protocol as described for dna but. In all cases i transfect a separate well for each sirna condition to test for transfection efficiency total of. Transfection protocol lipofectamine 3000 transfection reagent mor lung cancer cells complete growth medium component cat. Lipofectamine rnaimax transfection reagent delivery. The examples given within the protocol are for 96well plates, and table 1 provides the transfection reagent volumes for additional plate types. Optimization of the transfection condition generally, transfection optimization could be achieved by transfecting cells with sirnas targeting endogenous genes such as lamin ac and gapdh and then analyzing their expression by rtpcr or western blotting. The protocol indicated below is a reverse transfection protocol.
The protocal is from thermo fisher scientific important guidelines for transfection 1. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The highly efficient transfection reagent for mammalian cells. The following cell lines have been successfully transfected with the indicated transfection reagents. 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. Similarly, accuracy and reproducibility in preparing the plates for solidphase reverse transfection of sirnas are also critical. Transfection of crisprcas9 nuclease nls ribonucleoprotein. Rnaimax reverse transfections lipofectamine thermo fisher. Reverse transfection and forward transfection protocols can be used for most cell lines tested.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 proper culture techniques and procedures are an essential part of ensuring successful transfection. Invitrogen lipofectamine rnaimax transfection reagent 0. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. In animal cells, transfection is the preferred term as. Also prepare the sterile dna for transfection 2030 g10cm dish70 for a 15cm dish. Resuspend at 2 gml, and take the od on a small sample a 1. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. This protocol is for transfecting plasmid dna into mammalian cells. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. Transfection using effectene, lipofectamine, and polyfect reagents many reagents are now available for optimization of cell transfection efficiency. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Invivotransfection, in vivo rnai, animal rnai, sirna delivery.
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