To transfect cells with sirna, follow the protocol as described for dna but. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human tcell leukemia cells atcc cat. A sample protocol is listed here for transfection experiments performed in 6well plates. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22.
We have tried many transfection methods in tcells, but none of them have given us adequate results. This protocol is for transfecting plasmid dna into mammalian cells. The commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. Xtremegene hp dna transfection reagent is a high performance reagent that transfects a broad range of eukaryotic cells, including insect cells and hardtotransfect cell lines e. Mar 26, 20 gene transfer to t lymphocytes has historically relied on retro and lentivirus, but recently transposonbased gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression.
Jurkat transfection by electroporation harvard university. 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. Transitjurkat transfection reagent research reagent products. Invitrogen lipofectamine transfection reagent protocols have been optimized for. Comparison of transfection efficiency of nonviral gene. Precipitate 5 pmol around 10 g plasmid dna in 100 l. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Refer to the cell lines database for a list of cell types successfully transfected.
Lipofectamine 2000 transfection protocol 10cm plates reagents. Invitrogen lipofectamine 2000 transfection reagent fisher. Stealthsirna transfection protocol lipofectamine 2000. Troubleshooting for transfection of the crisprcas9 using. Add 6l of lipofectamine2000 from biostores and 100 l of. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Prewarm 50ml of optimum stored in cold room at 4c 10min. Scaling up or down lipofectamine 3000 reagent transfections. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. 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.
Cellspecific transfection protocols thermo fisher scientific us. Optimized conditions for successful transfection of human. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Each cell line was tested for with the neon 24 optimization protocol. A highly efficient method of transfecting hep g2 cells. Unique formulation maximize transfection performance in jurkat cells and other hardtotransfect cells such as k562, raw 264. Electroporation of cas9 rnps in difficult cell lines jurkat, ipsc. Cells in a 6 well dish, plated and at 70% confluence. Jurkat, human tcell leukemia, plasmid dna, lipofectamine 3000, l3000008. Transfection guide overview of transfection methods promega. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Lipofectamine 2000 reagent protocol thermo fisher scientific. Lipofectamine 2000 reagent thermo fisher scientific.
Improved delivery of cas9 proteingrna complexes using. Optimizing plasmid dna transfections with lipofectamine 2000. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Lipofectamine 2000 dna transfection reagent protocol. High transfection efficiency of jurkat cells with the neon. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The following protocol was optimized with jurkat and k562 cells, but can be used as a. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. If anyone has an experience, can you share it with me. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Transient plasmid dna transfection protocol for jurkat cells per well. I need to transfect plasmid dna into jurkat cells, but it seems that these cells are hard to transfect. 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.
Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Whats your transfection protocol for lipofectamine. For example, using electroporation we achieved 94, 91 and 44 % indel production efficiencies in jurkat t cells, k562 and. Transient plasmid dna transfection into hela and hacat. Invitrogen lipofectamine 3000 transfection reagent. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Incubate the cells for 18 h, or until the following morning.
Xtremegene hp dna transfection reagent highperformance. Lipofectamine transfection of c2c12 cells bridges lab. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. N v i s i on io 20 transit certified transfection reagents. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates.
I use 24well plates for transfections in triplicates. 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. Comparative transfection efficiency of jetprime versus its main competitor. Invitrogen lipofectamine 2000 transfection reagent. An efficient low cost method for gene transfer to t. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Quick reference protocol, msds and certificate of analysis available at.
What is the best method to transfect tcells jurkat. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. Highest transfection efficiency in many cell types and formats e. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Transfection protocol lipofectamine 3000 transfection reagent gct. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Invitrogen transfection reagent, recommended transfection sku, protocol.
The transfection efficiencies of the pte cells were 5% effectene. Rapid and highly efficient mammalian cell engineering via cas9. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Rapid and highly efficient mammalian cell engineering via. Quick reference protocol, msds and certificate of analysis available at mirusbio. The following morning, carefully aspirate the media. Transient plasmid dna transfection into hela and hacat cells. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Transfection of cultured eukaryotic cells using cationic lipid.
High efficiency delivery achieve 510% transfection efficiency in jurkat cells to ensure experimental success. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Lipofectamine 3000 reagent transfection efficiency. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Higher densities can lead to spontaneous differentiation.
Transfection protocol lipofectamine 3000 transfection reagent gct lung cancer cells complete growth medium component cat. I tried 6 well plates and 12 well plates all reagents scaled, of course. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Cationic lipidmediated transfection of adherent mammalian cells with dna. Luciferase activity was measured after 24 hours figures 24. As a biomaterial, peptides including tat 17, antennapedia homedomain peptide 18, and transportan peptide 19 were tested for gene delivery into cells with some promising in vitro results.
Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Transfection was measured by luciferase activity using a conventional assay. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Effective transfection of cas9 mrna or rnps using lipofectamine. Aims we herein report a convenient and affordable method based on in. For additional information on scaling your transfection reaction, see page 4. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The mechanism of lipofectamine 2000 mediated transmembrane. It is possible to plate and transfect the cells on the same day, but cells must adhere to. The highest transfection efficiency in many cell types and formats e. Transfecting the hardtotransfect lymphomaleukemia cells. Transfer of expression cassettes to t lymphocytes remains challenging, being based mainly on commercial kits. Apr 10, 2012 however, transfection rates of lipofectamine with plasmid dnas in the hardtotransfect lymphomaleukemia cells are disappointing 1216. 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. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene delivery methods such. Plate cells so they will be 7090% confluent at the time of transfection. Transfection and genome engineering thermo fisher scientific. A reverse transfection protocol was used to transfect mcf7 and. Using lipofectaminez 2000 for transfection provides the following advantages.
In biontex experiments gfp positivity increased to 90. Maintain p19 cells undifferentiated in mem with 10% serum 7. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Help on transfecting mcf7 and hct 116 cells transfection. Lipofectamine 2000 transfection reagent dartmouth college. To transfect cells with sirna, follow the protocol as. Genscript recommends using lipofectamine 2000 for all transfections. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Jurkat transfection by electroporation odified, j exp med.
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