Invitrogen lipofectamine 2000 transfection reagent. Cho transfection protocol, research methods and applications. The next generation in lipid technology pdf 4kb english format. Electroporation protocol for neon transfection system. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg.
Do not need to plate out cells one day in advance before actual transfection. Cotransfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Use the table below to find the right product for your experiment. 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. The following protocol is a guideline for transfecting approximately. Our laboratory uses pei over other cell transfection reagents because of its low cost. Reduction of thiolmodified oligonucleotides 575 kb reduction of thiol. Reverse transfection protocol for dna in 96well plates using transit2020 transfection reagent before you start. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. An electroporation protocol for efficient dna transfection in pc12 cells.
Many cell lines can be transfected successfully with pei but in our experience these two cell lines express the highest level of protein compared to other cells. While these protocols may serve as a helpful starting point for electroporation of other cell types as well, further optimization will be. Efficiencyup to 90% transfection and geneediting efficiency in extremely difficult cells, including immune, primary and stem cells. The device is preprogrammed with a 24well optimization protocol and supports a database to store up to 50 userspecified protocols. This website uses cookies so that we can provide you with the best user experience possible.
Superior performance for co transfection of sirna and plasmid dna. Highest transfection efficiency in cells at 90% confluency. How can i increase shrna transfection efficiency in hek293t. General protocol for transfection of stem cells, primary cells, and continuous cell lines with atcc genexplus transfection reagent. Deaedextran transfection is one of the oldest chemical, nonviral methods developed to transfer rna or dna to cultured mammalian cells 1, 2.
This protocol is appropriate for two suspension cell lines, chos and hek 293 gnti. Establishing stable cell lines there are many different approaches for establishing stable cell lines, depending on the type of expression youre interested in inducible vs. 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. 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. Guideit recombinant cas9 electroporationready user manual. 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. Transfection the transfer of genetic material into mammalian cells is a valuable tool that allows scientists to genetically manipulate neurons and neuronal tissue. Successful transfection of each cell type requires optimization of the basic protocol. A key step of any crispr workflow is transfecting the grna and cas9 into the target cells, but different transfection protocols have different advantages. L transfection of sirna to transfect cells with sirna, follow the protocol as described for dna but. Polyethylenimine pei transfection protocol virushost. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. The common causes of transfection failure are decrease transfection efficiency and decreased cell viability.
Here we provide protocols for performing knockout and knockin experiments in hips cells and cd34positive stem cells using the neon transfection system. The neon transfection system enables fast and efficient delivery of nucleic. Rnaimax transfection for huvec lipofectamine sirna plasmid cotransfection protocol with lipofectamine 2000 stealthsirna transfections oligofectamine stealthsirna transfection protocol lipofectamine 2000 transfecting plasmid dna into a549 cells using lipofectamine ltx reagent. Somebody would have a protocol of neurosphere transfection to share. Transfection and genome engineering fisher scientific. Early transfection studies used viral rna and dna, which were easy to propagate and purify, and allowed phenotypic discrimination of the transfected mammalian cells.
There are 3 options to select an electroporation protocol for your cell type. Transfection protocols thermo fisher scientific in. Targeted mutagenesis in wheat microspores using crispr. Deaedextran transfection of mammalian cultured cells. Use complete growth medium without antibiotics and antimycotics to plate cells for transfection. Co transfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. Pdf an electroporation protocol for efficient dna transfection in. Many cell types require unique protocols for optimal transfection. In addition, we discuss various transfection agents available from promega as well as general protocols for transfection and specific examples using our transfection reagents. Lipofectamine transfection of c2c12 cells bridges lab protocols.
Plate cells at an appropriate density so they will reach 7080% confluence at the time of transfection plating media should contain 1020% serum. The neon transfection system uses a single transfection kit neon kit that is compatible with various mammalian cell types including primary and stem cells thereby avoiding the need to determine an optimal buffer for each cell type. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. General protocol for transfection of stem cells, primary. 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.
Moremen lab, 121511 transient transfection of hek293f suspension cultures using pei cells are grown in suspension on a platform shaker in a humidified 37c co. The neon transfection system offers open and transparent protocols that are optimized for ease of use. Assays should be done immediately following 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. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. 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. Transfection viability tip type 2,150 20 1 2 x 107 23% 95% 10 l peripheral blood mononuclear cells pbmcs were transfected using the neon transfection system and 1. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. If you choose not to optimize transfection parameters, use the general conditions recommended below. Ar42j is a pancreatic cancer cell line that has proven to be a suitable host for transfection.
Rnaimax transfection for huvec lipofectamine sirna plasmid co transfection protocol with lipofectamine 2000 stealthsirna transfections oligofectamine stealthsirna transfection protocol lipofectamine 2000 transfecting plasmid dna into a549 cells using lipofectamine ltx reagent. En attractene transfection reagent handbook qiagen. This protocol describes the delivery of a cas9 ribonucleoprotein rnp complex. Conclusions there are many options for the transfection of stem cells, each with tradeoffs in regards to performance, cell viability, ease of use, and cost.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. Amaxa 4dnucleofector optimization protocol for cell lines. Biochemistry, molecular biology, and cell biology protocols cell transfection protocol calcium phosphate method based on maniatis. The protocol for lipofectamine messengermax reagent is straightforward, requiring the same basic steps as protocols for dna transfection reagents, and we offer a positive control gfp mrna that can be used to evaluate the system. Get improved transfection outcomes with the neon transfection system. I use 24well plates for transfections in triplicates. Any other transfection protocol may be adapted in a similar way to the handling in ibidi slides. Instructions for using the neon transfection system for transfection of mammalian cells are described below. However, the neon transfection system with these conditions can also be used for standard delivery of dna into pbmcs. 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.
Using the neon transfection system refer to the manual for details on setting up the neon device and neon pipette station. We compared two electroporation systems neon transfection and gene pulser xcell and three chemical transfection methods lipofectamine. By comparing efficiencies from replicate experiments we proved electroporation in particular neon to be the method of choice. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Transient mammalian cell transfection with polyethylenimine pei.
The following protocol is given for transfection in 24well plates, refer to table 2 for transfection in other culture formats. Protocol for cell transfection in ibidi slides due to the broad spectrum of transfection methods, this application note only describes an example of dna transfection conducted in slides vi 0. Transfection conditions should be optimized for each cell type to ensure. Altr crisprcpf1rnp electroporation, neon transfection system 496 kb altr genome editing detection kit 910 kb altr genome editing detection kit.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Superior performance for cotransfection of sirna and plasmid dna. Ar42j transfection reagent pancreatic carcinoma altogen. User guides and protocols integrated dna technologies.
Universal transfection reagent protocol sigmaaldrich. 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 is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. How can i increase shrna transfection efficiency in hek293t cells. Kenji yamato at tokyo medical and dental university protocol 1. Lipofectamine 2000 reagent thermo fisher scientific.
Use table 1 to select the reagent volumes for various plate sizes. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. 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. Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Variables to consider for optimization include, but are not limited to, cell density, transfection reagent, duration of transfection, and dna concentration. It may also refer to other methods and cell types, although other terms are often preferred. Transient transfection of cos1 cells upstate this protocol has been optimized for cos1 cells. Quick howto video on how to use and conduct the optimization protocols for the neon transfection system for electroporation instrument. Protocol i transient transfection e3314 introduction. The total volume of transfection complex medium, dna and viafect transfection reagent to add per well of a 96well plate is 510. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
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. 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. Application note gene delivery solutions stem cell. Pei is the basis of most commercially available transfection agents and acts as a very cost effective transfection vector. For a transient transfection follow protocol 1 except you dont have to cut the plasmid and do not add any selecting antibiotic. 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. 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. Use complete growth medium without antibiotics and. 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. This protocol is specific for the establishment of cell lines that constitutively express gfptagged proteins. A sample protocol is listed here for transfection experiments performed in 6well plates. The unique properties of the delicate cells make transfection challenging and necessitate specialized techniques. In 1957, the original cho cell line was derived by theodore t.
1304 1537 638 104 1099 176 1407 1387 277 806 839 1412 1085 953 429 323 127 1120 807 564 48 108 1423 127 1529 79 267 474 157 945 458 35 893 546 981 1203 1095 104 103 976 1088 129 1322 1445