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. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. The protocol indicated below is a reverse transfection protocol. 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 cell database sample to insight qiagen. Current transfection techniques can be broadly divided into viral, chemical, and physical methods. Transfection protocols thermo fisher scientific us. An efficient transfection reagent, such as the ar42j transfection kit for pancreatic carcinoma cells, offered by altogen biosystems, can improve the outcome of this research.
As always, qiagen cannot verify data supplied from outside sources. Does anyone have a good transfection protocol by using. While the application of bacterial transformation is typically for overexpression of genes in an effort to produce large quantities of proteins that can be purified and studied, dna transfection is typically used for the studying the effect of gene expression in a. Assays should be done immediately following transfection. Rnai transfection protocols thermo fisher scientific us. As an example, we will use antihnrnp a1 and antiigg antibodies. This approach can be adapted for different cell lines and different transfection reagents. Cellspecific transfection protocols thermo fisher scientific us. I begin procedure by seeding the cells in a 6 well plate at a 1x105 cellsml density, letting.
Also calculates the volumes needed for transfection protocols based on well. Transfection guide overview of transfection methods promega. Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the. Expression of egfp allowed visualisation of transfection efficiency following different incubatio. Subculturing, also referred to as passaging, is the removal of medium and transfer of cells from a culture into fresh growth medium, in order to propagate the cells. In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. Brightfield image of llc1 cells in culture, prior to transfection. However, the 4dnucleofector tm y unit offers the opportunity to keep cells in adherence during electroporation. Fugene hd transfection reagent lets you work in your cancer model. Warm up growth media and trypsin in a 37c water bath 30 minutes prior to the start of the experiment 30 minutes 2.
If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Cotransfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. In addition, our knowledge database contains transfection data for close to 1500 cell types. Transfection protocol calculator our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Optimisation of the transient transfection protocol and openi. A few applications that popularly utilize co transfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. For example, one reagent may work well with hek293 cells, but another reagent may be a better choice when using hepg2 cells.
Promega provides the fugene hd protocol database to help identify a protocol for your cell line when using the fugene hd 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. For a transient transfection follow protocol 1 except you dont have to cut the plasmid and do not add any selecting antibiotic. To download a qiagen transfection protocol for your cell type, nucleic acid, and plate format of interest, visit the transfect protocol database. Transfecting plasmid dna into a549 cells using lipofectamine. A dropdown menu allows you to search the database by cell line, plate type and number of cells to be transfected. Transfection conditions should be optimized for each cell type to ensure. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Ar42j is a pancreatic cancer cell line that has proven to be a suitable host for transfection. To simplify the transfection optimization process, we have created a transfection selection tool that provides recommended transfection reagent with optimized.
The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. 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. General protocol for transfection of stem cells, primary cells, and continuous cell lines with atcc genexplus transfection reagent. These protocols were developed by promega corporation or fugent, l. The transfection experts bring you the reagent agent a tool designed to help you determine the best delivery solution for any nucleic acid into any cell type. If you find this doesnt work for your specific cell type, then you can look to our cellspecific protocols. Transfection of neural stem cells with lipofectamine stem transfection reagent in stempro medium transfecting plasmid dna into calu1 cells using lipofectamine 3000 reagent transfecting plasmid dna into calu6 cells using lipofectamine 3000 reagent. Transfection conditions should be optimized for each cell type to ensure maximal gene expression with minimal cellular toxicity. We specifically use this protocol with lentix 293t cells, a cell line optimized for production of lentiviral vectors. 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. As the name suggests, this method utilizes viruses to deliver genetic materials into cells. Count the cells using standard trypan blue exclusion. Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity.
Use the fugene hd protocol database to see if transfection conditions for your cell line are available. Plate cells for transfection only 1 day before the experiment. Universal transfection reagent protocol sigmaaldrich. The fugene hd protocol database is a collection of protocols that guide the transfection process for a variety of cell lines. Ar42j transfection reagent pancreatic carcinoma altogen. 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.
Eplate 16 preparation and background measurement 15 minutes a. Protocol i transient transfection e3314 introduction. General protocol for transfection of stem cells, primary. I have been trying to use the crisprcas9 system in u373 cells using pei chemical for transfection. This process may involve choosing a new transfection reagent. Adherent primary cells, especially neurons, at defined developmental stages can be transfected using the y unit without affecting their functionality and. Use complete growth medium without antibiotics and antimycotics to plate cells for transfection. Does anyone have a good transfection protocol by using lipofectamin 3000. It may also refer to other methods and cell types, although other terms are often preferred. A collection of interactive web tools and downloadable software to help you do.
The transfect protocol database is an invaluable resource for transfection experiments which provides transfection protocols for specific cell types and plate formats. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagentdna complex. All available information is shown in the detailed view. Many researchers conduct transfection experiments on a daily basis, which requires a concise understanding of the biological background, precise and detailed planning, and a robust transfection protocol. A sample protocol is listed here for transfection experiments performed in 6well plates.
Rnai transfection protocols thermo fisher scientific ng. Application protocol reverse transfection of sirna on eplates. Use complete growth medium without antibiotics and. The following protocol is given for transfection in 24well plates, refer to table 2 for transfection in other culture formats. Rna interference rnai is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. A commonly used control is the same transfection reagent plus a noncoding rnai. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection method, protocol, and technique, invivo and invitro reagents, stable and transient transfection, rnai, sirna gene silencing, in vivo transfection, rnai in animals, library screening, cancer cell lines, liposomes. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Transient transfection and coimmunoprecipitation procotol. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. Nonviral in vivo delivery is a safe method for delivery of nucleic acids in different organs of various animal models. After protocol optimization, the results can be easily reproduced.
Kenji yamato at tokyo medical and dental university protocol 1. This database is by no means exhaustive, but a collection of data dependent on the efforts of our technical service team. Dna transfection is the process by which dna is taken up by eukaryotic cells. Transfection guide overview of transfection methods. As a drawback, transfection by cationic polymers has a very low efficiency qiagen cannot verify data supplied from outside sources. Use the table below to find the right product for your experiment. For easy quantification of transfection efficiency we used antihnrnp a1 antibodies labelled with atto550nhs and fitclabeled igg. 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. Invitrogen lipofectamine transfection reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Xtremegene transfection database 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. To achieve effective silencing, rnai is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. Optimisation of the transient transfection protocol and reliability tests. The transfection efficiency of pegfpn1 or prfp was measured using a flow cytometer bd pharmingen, san jose, ca, usa, and data analysis was performed using flowjo software.
The goal of transfection is usually to investigate the effects of expressionsilencing of target genes. Co transfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. 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. Invitrogen has the most complete collection of transfection reagents with. Certain cell types are less permissive to in vivo transfection and require the development of specific transfection reagents to improve nucleic acid targeting efficiency. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics. 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. A few applications that popularly utilize cotransfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. Amaxa 4dnucleofector optimization protocol for cell lines. Transfection cell database our searchable database contains data from researchers like yourself who have shared their experimental results with us. Optimize transfection of cultured cells promega corporation.
Antibody transfection into neurons as a tool to study disease. Transient transfection and coimmunoprecipitation protocol. How to optimize your transfection using the nucleofector technology when no cell typespecific protocol is available for your cell type of interest. The database includes over 25 cancer models and provides transfection protocols specific for each cell line.
General transfection protocols thermo fisher scientific us. This website uses cookies so that we can provide you with the best user experience possible. 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. Electroporation usually requires cells to be in suspension for transfection.
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