For plate size volume of sirna transfection reagent per well l reactions 1. It is not necessary to remove complexes or changeadd. Use highquality plasmid dna that is free of proteins, rna, and chemicals for transfections. Depending on the cell line and cell density the dharmafect. 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. Transfection efficiency was increased in a dose dependent way with saturation 30 g. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions. This cell line has been adapted for growth in suspension culture in serum free culture medium similar to the freestyle 293f cell line. Let stand 15 minutes at room temperature to allow purefectiondna complexes to form 5. This approach can be adapted for different cell lines and different transfection reagents. Test a variety of brands and lots of fbs to find one suitable with your protocols. This is often the best place to start, especially in a new cell line. Largescale transient transfection of suspension mammalian. Universal transfection reagent protocol sigmaaldrich.
Our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Simply select the cell type, nucleic acid, and culture format to receive a qiagen transfection protocol to print out or download in convenient pdf format. Assay for transposon removal in target cells 72hrs after transfection. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Prior to transfection, replace media with fresh antibiotic free growth. Qiagen protocols two types of protocol are provided in the transfect protocol database.
L volume of diluted dharmafect transfection reagent in serumfree medium. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Warm up growth media and trypsin in a 37c water bath 30 minutes prior to the start of the experiment 30 minutes 2. Promega corporaoon 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526. All products were used according to manufacturers protocol. For more information on using the antibody expressing positive control vector, see page 18.
Mission sirna transfection reagent s1452 technical bulletin. Transfection by electroporation pubmed central pmc. Lipofectamine 2000 reagent thermo fisher scientific. Viafect transfection reagent can be used in transfection protocols in the presence of serum, allowing transfection of cell types that require continuous exposure to serum, such. Transfection guide overview of transfection methods promega. This product is for laboratory research only and not for diagnostic use 2008 signagen laboratories the following protocol is given for transfection in 24well plates, refer to table 2 for transfection.
Transient transfection of serum free suspension hek 293 cell culture for efficient production of human rfviii. We specifically use this protocol with lentix 293t cells, a cell line optimized for production of lentiviral vectors. Transfection guide overview of transfection methods. Hek293 standard transfection protocol 24well plate.
Add dropwise to cells culture and swirl to disperse 6. Stable transfection viafect transfection reagent can be used to produce stable transfectants. However, we recommend optimizing transfection conditions using transient transfection studies prior to selecting for stable transfectants. Largescale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the.
Xtremegene transfection reagents from roche have been used for a wide range of applications and a vast array of cell lines, primary cells and stem cells. Use highly purified, sterile, and contaminantfree dna for transfection. Use complete growth medium without antibiotics and antimycotics to plate cells for transfection. Transfection of hek 293 ebna cells could be inhibited or completely diminished if such critical components were present at other steps of the process. Transfast transfection reagent technical bulletinpdf. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. See the protocols and applications guide chapter on dna purification for information about purifying transfectionquality dna. Some transfection protocols require serumfree conditions for optimal performance, since serum can interfere with many commercially available transfection reagents. Mix the 2 plasmidstogether before adding optimem serum free media co transfection upto 45 plasmids is very common and follow the protocol for lipo2000. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Calcium phosphate transfection optimization for serumfree suspension culture article pdf available in cytotechnology 353. See our reagent specific protocols for more details.
Same protocol as above except use a961 plasmid instead of a979 plasmid. Transfection is the introduction of dna, rna, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Crisprcas9 ko plasmid and hdr plasmid transfection this protocol is recommended for a single well from a 6well tissue culture plate. The transfection protocol has been published previously.
Maintain p19 cells undifferentiated in mem with 10% serum 7. I use 24well plates for transfections in triplicates. Plate cells so they will be 6080% confluent at the time of transfection. Protocol i transient transfection e3314 introduction. Pdf calcium phosphate transfection optimization for serum.
Use the indicated volume of dna and p3000 reagent with each of the two volumes of lipofectamine 3000 when. The pei transfection described in the standard pei transfection protocol typically. Plasmid dna preps that are endotoxinfree and have a260280 absorbance ratio of 1. Transfection efficiencies will vary with cell type can be used only for transient transfection typically produces less than 10% delivery in primary cells. On the plus side, you can use it to transfect large dna fragments and achieve good transfection efficiencies using cell lines. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Plate cells so they will be 7090% confluent at the time of transfection. Transfection methods are used for a range of applications, including gene function studies, modulation of gene expression, biochemical mapping, mutational analysis and production of recombinant. As for all transfection methods, electroporation has its advantages and disadvantages. Nov 24, 2011 the transfection protocol has been published previously. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. This article provides detailed protocols, sequences and strategies that allow the expression and purification of endotoxin free antibody reagents suitable. Polyjet reagent was formulated for dna transfection only.
As a guide, cells should be transfected at 4080% confluency. Fbs can be purchased already head inactivated or it can be inactivated in the lab by heating to 56. Transfection conditions should be optimized for each cell type to ensure. Protips different brands and lots of fbs can promote or inhibit transfection. Highquality dna free of nucleases, rna and chemicals is as important for successful transfection as the transfection reagent chosen. Transfection of cells with the betagalactosidase control 1. Make dnalipofectamine 3000 complexes in serum free. Transfection technologies the introduction of nucleic acids into cells is one of the most valuable and frequently used tools of biological science. I tried lipofectamine and attractene which both had a bad efficiency.
The basic protocol describes the electroporation of mammalian cells, including es cells for the generation of transgenic and knockoutin mice. Transient plasmid dna transfection protocol per well of a 6well plate a. See our sirna transfection reagent protocols for more details. Store small aliquots of the 1x stock solution at 20c for up to 6 months. The protocal is from thermo fisher scientific important guidelines for transfection 1.
University college cork, ireland request a free sample transfection is the process by which nucleic acids are introduced into mammalian cells. Sfm complexation medium is a serum free, animal origin free. Transfection protocols often require serum free conditions for optimal performance because serum can interfere with many commercially available transfection reagents chan et al. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses.
Approximately 1824 hours before transfection, plate cells in 2. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Mission sirna transfection reagent catalog number s1452. If you find this doesnt work for your specific cell type, then you can you look to our cell specific protocols for further optimization. Transient transfection of serumfree suspension hek 293. At the last minute, adjust ph of the transfection buffer to 7. Lipofectamine 3000 transfection reagent protocol transfect cells according to the following table. Pei transfection of 293 cells california institute of. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Prepare transfection complexes by mixing 40 l of serum. Transient transfection of hek293f suspension cultures using pei. A sample protocol is listed here for transfection experiments performed in 6well plates. Please adjust all volumes according to your optimal transfection scale. Transfection of pluripotent stem cells with lipofectamine. 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. Higher densities can lead to spontaneous differentiation. Lipofectamine 3000 reagent protocol lipofectamine 3000. Transfection protocol for adherent cells follow this protocol as a starting point, and further optimize the experiments using the guidelines given. The transfection protocol online library contains protocols obtained from the literature, developed by biorad scientists, or submitted by scientists like you.
The optimal transfection conditions for a majority of adherent cell lines are given in the standard protocol described below. Transient transfection of serumfree suspension hek 293 cell. So, we used 30 g per sample in the final protocol, but of course it can be increased e. General protocol for transfection of stem cells, primary cells, and continuous cell lines with atcc genexplus transfection reagent. Fast and easy protocol is compatible with serum, serum free medium and antibiotics. Mission sirna transfection reagent s1452 technical. It may also refer to other methods and cell types, although other terms are often preferred. In conclusion these data demonstrated that a transfection protocol can be as critical to efficient pei mediated transient transfection of hek 293 ebna cells as the transfection medium. In all cases i transfect a separate well for each sirna condition to test for transfection efficiency total of 4 wells per sirna. Transfection protocols often require serum free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. Adjust cell and reagent amounts proportionately for wells or dishes. This website uses cookies so that we can provide you with the best user experience possible. Transfection conditions should be optimized for each cell type to ensure maximal gene expression with minimal cellular toxicity.
Transfection protocols thermo fisher scientific in. Add 22 l of 25mm chloroquine to each 15cm dish for 10cm plate 7. Genejuice transfection reagent is a proprietary formulation optimized for maximal transfection efficiency, ease of use, and minimal cytotoxicity for mammalian cells. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. General protocol for transfection of stem cells, primary. Transit lt1 transfection reagent protocol for mir 2300, 2304, 2305, 2306 page 3 of 6 plasmid dna transfection protocol the following procedure describes how to perform plasmid dna transfections using. Eplate 16 preparation and background measurement 15 minutes a.
Transfection of cells with a protein of interest the following protocol describes the transfection of protein into one well of a 6well plate. Browse protocols to view our library and find your starting point or submit a protocol by clicking the proper technology. Transfection mix serum free medium volume of complete medium. Universal and efficient electroporation protocol for genetic. Attractene transfection reagent and nanofect transfection reagent. Plasmid dna for transfections should be free of protein, rna and chemical contamination a260. Jun, 2010 transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells.
Use the table below to find the right product for your experiment. Except for cases where the basic protocol is absolutely right, we suggest reproducing the initial findings and creating stronger data with a few optimization steps. Precultures underwent passages every 23 days to keep the cells in their exponential growth phase. The alternate protocol outlines modifications for preparation and transfection of plant protoplasts. Some transfection protocols require serum free conditions for optimal performance, since serum can interfere with many commercially available transfection reagents. Transfection protocol lipofectamine stem transfection reagent transfection of pluripotent stem cells with lipofectamine stem transfection reagent in essential 8 medium psc growth medium, passaging reagents, and complexation medium component cat. Animalfree polycationic liposomal transfection reagent optimized for. This is one of four transfection protocols you can find in this web page set.
The following protocol is given for transfection in 24well plates, refer to table 1 for transfection in other culture formats. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. The cells should be transfected within 510 of adding the chloroquine. Pei transfection of 293 cells raav production by triple transient transfection of 293ft cells i. Application protocol reverse transfection of sirna on eplates. Whether this is your first transfection or youre a seasoned pro, our new protocols and support tools help you achieve truly effortless, successful transfection experiments. Lipofectamine 2000 dna transfection reagent protocol. Cell density, amount of transfection complex, viromerpdna or mrna ratio, and duration of transfection incubation time are the determining factors for protocol optimization. Make dnalipofectamine 3000 complexes in serumfree medium.
Reverse transfection protocol acea biosciences inc. Kenji yamato at tokyo medical and dental university protocol 1. All transfections were performed in serumfree media for four hours followed by complete media. To transfect cells with sirna, follow the protocol as. Expi293 expression system user guide for scalable transfection of expi293f cell lines in a chemically defined, serum free medium, using the expifectamine 293 transfection kit for scalable metabolic protein labeling using expi293f cell lines, in a chemically defined, serum free, methionine free medium using a. 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 sections of the website you find most interesting and useful. Plasmid dna for transfections should be free of protein, rna and chemical contamination a260a280 ratio of 1. A few hours before transfection, the cell suspension was centrifuged 300 g for 5 min and resuspended in fresh medium at 5. The in vivo protocols describe the use of electroporation to deliver plasmid dna to muscle and skin.
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