For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. How to increase transfection efficiency when lipofectin. How can i optimize the transfection efficiency using lipofectamine. 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. Because of the low infection efficiency, i am a bit worried the same thing will happen on transient transfection using lipofectamine 2000 on nih3t3. 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. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Protocols for transfecting the most common cell lines with x. I used lipofectamine 2000, 3000, pei, fugene 6 and hd, electroporation and no transfected cell. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Tm328 fugene hd transfection reagent technical manual. The proprietary formulation of lipids and polymers ensures the highest possible transfection efficiencies and viabilities for nih3t3 cells. Higher densities can lead to spontaneous differentiation. Enhanced and simple transfection protocol for a variety of cell lines.
Follow these important guidelines when transfecting. The amount of protein and the incubation time with the transfection complex mixture used may be adjusted for optimal transfection efficiency. Does someone have successful transfection in 3t3l1 cells. Our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. For additional information on scaling your transfection reaction, see page 4. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Have you even transfected nih3t3 fibroblasts using pei reagent. Assay measure protein expression introduction this protocol shows optimum transfection condition using hilymax in nih3t3 cells. Hela f5 cells transfected with f517 oligo, either 1x or 2x dna.
Prepare transfection complexes by mixing 40 l of serum. 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. Metafectene pro, metafectene, fugene, lipofectamine 2000 experimental procedures transfection protocol. At 72 hours posttransfection the cells were analyzed by western blot for protein expression levels normalized by total protein, 10 g of total protein loaded per each well. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Transfection of nih3t3 cells with lipofectamine 2000. Transfecting plasmid dna into nih3t3 cells using lipofectamine. Transfecting sirna using lipofectamine rnaimax pin.
Make sure and evenly distribute the cells on the coverslips by swirling plate. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Xgal staining of nih3t3 cells 24 hours after transfection by fugenehd, fugene6 and competitor lipofectamine 2000, 5,000 cellswell in 96 well plate click here to view the protocol fugenehd. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Have somebody done the transfection in 3t3 l1 cell lines. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Materials free fulltext nanomagnetic gene transfection for non. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of.
Transfections with lipofectamine 2000 herein named l2k. Comparison of transfection efficiency between commercialy. Optimized conditions for successful transfection of human. Transfection guide overview of transfection methods promega. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Dna plasmid expressing gapdh or sirna targeting gapdh were transfected into nih3t3 cells following altogen biosystems transfection protocol. To tranfect nih3t3 cells in 24well plate, follow optimum condition for transfection and transfection procedure. Comparative transfection efficiency of jetprime versus its main competitor. I want to try to use hela cell and lipofectamine 2000 to do.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. For using lipofectamine 2000 reagent, the above plasmids were. 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. Change the growth medium 4 hours after transfection. Nih3t3 transfection kit swiss mouse fibroblasts altogen. The 3t3 cell line was originally established from a swiss albino mouse embryo tissue in 1962 by two scientists at new york university school of medicine. Lipofectamine 2000 dna transfection reagent protocol. Follow these important guidelines when transfecting dna into nih3t3 cells using lipofectamine ltx reagent. Highest transfection efficiency in many cell types and formats e. This is often the best place to start, especially in a new cell line. I have performed lipofectamine transfection of cdna3.
Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates. Nih3t3 is commonly used for in vitro studies, so it gives me the perception its easy to transfect and infect. Transfection protocol lipofectamine 3000 transfection reagent. Xgal staining of nih3t3 24 hours after transfection fugene hd. Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1.
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. How to increase transfection efficiency when lipofectin 2000. Some of my cells will dried if i used the volume suggested by the manual. Transfecting stealth rnai or sirna into 3t3 l1 cells. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Invitrogen lipofectamine 3000 transfection reagent 0. Nih3t3 cell avalanche transfection reagent is a new, proprietary solution specifically designed for transfection on nih3t3 cells. Few cells were transfected by lipofectamine plus but it was not too good. Genscript recommends using lipofectamine 2000 for all transfections. Transfection protocols thermo fisher scientific in. Lipofectamine 2000 reagent thermo fisher scientific.
Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. A sample protocol is listed here for transfection experiments performed in 6well plates. Cells are dying after transfection due to toxicity of either dna lipofectamine reagent. Nih3t3 standard transfection protocol 24well plate. The magnefectnano system was evaluated for transfection efficiency, and any potential. Lipofectamine ltx transfection reagent from invitrogen. An improved method for increasing the efficiency of gene. Fugener hd transfection reagent technical manual tm328. If you find this doesnt work for your specific cell type, then you can you look to our cell specific protocols for further optimization.
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. Here we compared the transfection and viral transduction efficiency using. Sep 25, 2007 invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. Transpass p transfection reagent should be stored at 4c. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Each reagent was used to transfect hek 293, hela, lncap, a549, and.
Hela transfection is usually pretty standard in terms of protocols just make sure. The fluorescentlabeled control protein lgg labeled with a. Using lipofectaminez 2000 for transfection provides the following advantages. Nih3t3 cell avalanche transfection reagent,will be provided. Fast and efficient transfection of mouse embryonic stem cells. The highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Add 6l of lipofectamine2000 from biostores and 100 l of.
The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. Follow these important guidelines when transfecting stealth rna or sirna into 3t3 l1 cells using lipofectamine 2000. Lipofectamine transient transfection of cos7 cells to analyze. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency.
Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. Maintain p19 cells undifferentiated in mem with 10% serum 7. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. How to increase transfection efficiency when lipofectin 2000 was used on the cell line 3t3 or neuronal cell line gt17. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Have you even transfected nih3t3 fibroblasts using pei. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. To transfect cells with sirna, follow the protocol as described for dna but. In addition metafectene pro was also used at 1 g plasmid and 4 l reagent 1. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I use 24well plates for transfections in triplicates.
Protocol for lipofectamine transfection in nih3t3 cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 transfection reagent from invitrogen. I want to try to use hela cell and lipofectamine 2000 to do transfection. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
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