Generally need to have 2 g of dna for each tranfection. Systematic screening of commonly used commercial transfection. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. Transfection is a powerful tool for analyzing the roles of certain genes. Lipofectamine rnaimax reagent protocol thermo fisher scientific. Reverse transfection magnetofectamine protocol v 1. Transfection protocol lipofectamine 3000 transfection reagent gct. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Has anyone here attempted reverse transfection using. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Transfection protocol lipofectamine 3000 transfection. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Comparative transfection efficiency of jetprime versus its main competitor. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
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. Reverse transfection on cell arrays for high content. A b 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. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Genscript recommends using lipofectamine 2000 for all transfections. Transfecting sirna using lipofectamine rnaimax pin. Luciferase activity was measured after 24 hours figures 24. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from. Invitrogen lipofectamine 2000 transfection reagent. Cells are added directly to the lipofectamine 2000rna complexes and transfection occurs while cells are attaching to the well. Optimized delivery of sirna into 3d tumor spheroid cultures. Lipofectamine rnaimax is a proprietary formulation specifically developed for.
Do any one has protocol for stable transfection with lipofectamine 2000. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfection protocol lipofectamine 3000 transfection reagent.
For reverse transfection, cells in optimal growth phase are prepared so that it is possible to seed 2. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Transfection was performed as described in protocol 2 with the following modifications. Forward and reverse transfection protocols mirus bio. A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. 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.
The image below figure 1 is an example of the microarrays that can be created with this approach. Lipofectamine 2000 transfection protocol 10cm plates reagents. Add 6l of lipofectamine2000 from biostores and 100 l of. I am about to start my stable transfections with lipofectamine 2000. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 reagent thermo fisher scientific. Our lab uses lipofectamine 3000 for transfections, but the only protocols for reverse transfection i can find on thermofisher scientifics website deal with their rnaimax reagent.
Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. We optimized conditions for reverse transfection with the hek 293t cell line. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. In the protocol described here, we use a pool of three sirna duplexes per target gene per. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.
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. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Highthroughput parallel transfection techniques are a prerequisite to perform largescale experiments, such as genomewide rnai screens, in mammalian tissue culture cells 1,2,3,4. We call this approach reverse transfection and we have developed two methods to reverse transfect cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Prewarm 50ml of optimum stored in cold room at 4c 10min. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. The term reverse transfection comes from the invention and development of a microarraydriven gene expression system by junald ziauddin and david m. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. General guidelines for transfection follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells.
Transfection reagents product protocols thermo fisher. Comparison of reverse transfection efficiency of lipofectamin 2000 with metafecten pro. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Reverse transfection combines rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 transfection protocols. 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.
May 21, 2018 transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best performing polymeric nanoparticles transfected 57. We have found that effectene, lipofectamine 2000 and fugene 6 work with this protocol. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. This reference provides general guidelines and a procedure to cotransfect plasmid dna and an rnai molecule i. In short, a different rnai molecule is put in each well prior to transfection and combined with diluted lipofectamine 2000 to form complexes in each well. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Today, several ways of introducing nucleic acid into cells are described. 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 lipofectamine 3000 transfection reagent. A sample protocol is listed here for transfection experiments performed in 6well plates. Rnaimax reverse transfections lipofectamine thermo. In the present study, we screened a large set of transfection reagents using relatively easy and hardtotransfectcelllines and identi.
Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. Optimized conditions for successful transfection of human. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Reverse transfection of stealth rnai or sirna using. Reverse transfection of plasmid dna automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in highthroughput assays. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3.
Max % of positive cells after transfection of a gfpplasmid 3. 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. The protocal is from thermo fisher scientific important guidelines for transfection 1. For suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. Scaling up or down lipofectamine 3000 reagent transfections. 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. What percentage confluency should be used for stable transfection. Refer to the cell lines database for a list of cell types successfully. Highest transfection efficiency in many cell types and formats e. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Use this procedure to reverse transfect stealth rnai or sirna into.
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. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Improved delivery of cas9 proteingrna complexes using. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Also, is the nucleic acid in the media responsible for cellcell clumping. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Cotransfecting rnai and dna with lipofectamine 2000. Photograph of expected results dnalipofectamine scaling up or down. Lipofectamine 2000 transfection reagent dartmouth college. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. In this, the positively charged deaed binds to the negatively charged dna. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Comparative study of metafecten pro and lipofectamine 2000 by. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Advanced transfection with lipofectamine 2000 reagent. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Reverse transfection cell microarray rtcm is a good method for parallel high throughput analyzes of gene. Highthroughput parallel transfection techniques are a prerequisite to perform largescale experiments, such as genomewide rnai screens, in mammalian. Lipofectamine 2000 transfection reagent from invitrogen. One of the oldest methods is transfection using diethylaminoethyldextran deaed. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension.
Stealth rnai, sirna, shrna plasmid or mirna plasmid into mammalian cells using lipofectamine 2000 reagent lipofectamine 2000 reverse transfection. The two most powerful transfection technologies in one kit. Other reagents did not demonstrate effective transfection to this cell line. The efficiency of transfection is somewhat lower, but probably can be improved by optimizing protocols for this reagent.
I use 24well plates for transfections in triplicates. An invitrogen guide mentioning lipofectamine 2000 suggests using reverse transfectin for stealth rnai or rnai. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. As dna are printed on a glass slide for transfection process to occur before the addition of adherent cells, the order of addition of dna and adherent cells is a reverse of that. A typical forward transfection protocol using transit2020 transfection reagent can be found here. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. This protocol has been used successfully with the cells and conditions outlined below.
Highthroughput applications, such as knockdown studies or target screenings, often include cell transfection. If so, how could i prevent it from happening when doing a reverse transfection. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Lipofectamine 2000 was also reported in u2os cells. Reverse transfection an overview sciencedirect topics.
Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. For additional information on scaling your transfection reaction, see page 4. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Rnaimax reverse transfections lipofectamine thermo fisher. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Thee two most important factors in choosing transfecting reagent are. 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. The efficiency of transfection is somewhat lower, but probably. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
1507 46 323 340 1176 1076 525 1477 661 1383 396 1470 1007 1470 1506 70 1159 777 1469 1228 776 1034 973 1022 155 763 5 16 309 587 1263 373 439 1455 817