I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Lipofectamine 3000, and p3000 enhancer as suggested in the manual for. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. It may also refer to other methods and cell types, although other terms are often preferred.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Invitrogen lipofectamine 3000 transfection reagent fisher. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. However, the electroporation methods enable efficient transfer of.
B hela cells were transfected with luciferase encoding plasmid dna using either transit. Invitrogen lipofectamine 2000 transfection reagent. 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. Prior to flow cytometry, visualize cells under a brightfield microscope to verify dissociation following incubation with tryple reagent. Refer to the cell lines database for a list of cell types successfully. It is a relatively modern method first described by felgner et al. Transitx2 transfection reagent enables superior transfection in a variety of cell types. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Concentration of dna for stable transfection with lipofectamine 2000 and the per transfection and transduction. 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.
Transfection efficiencies lower than 50% were not reported in this table. The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. The transfection efficiencies of the pte cells were 5% effectene. Prior to flow cytometry, visualize cells under a brightfield microscope to verify dissociation following incubation with.
Optimized conditions for successful transfection of human. The ability to efficiently transfer dna or small interfering rna sirna is. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent. Generally need to have 2 g of dna for each tranfection. Lipofectamine 2000 transfection reagent dartmouth college. Can i use lipofectamine 2000 to cotransfect plasmids and sirna. Transfection was measured by luciferase activity using a conventional assay. Max % of positive cells after transfection of a gfpplasmid 3. Does anyone transfected u251, a172 gbm cell lines with. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfecting a human neuroblastoma cell line with monster. Lipofectamine 2000 reagent thermo fisher scientific. 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.
Stealthsirna transfection protocol lipofectamine 2000. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Transfection protocol lipofectamine 3000 transfection reagent. Transfection guide overview of transfection methods promega. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Lipofectamine 2000 transfection reagent from invitrogen. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. What percentage confluency should be used for stable transfection. How many injections can i perform with the invitrogen invivofectamine 3. Lipofectamine 2000 for transfection provides the following advantages.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. I want to try to use hela cell and lipofectamine 2000 to. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. How does transfection with lipofectamine 2000 work. Transfection guide overview of transfection methods. Genscript recommends using lipofectamine 2000 for all transfections.
Using lipofectaminez 2000 for transfection provides the following advantages. Lipofection has become more common than the capo 4 precipitation, because it is just as simple to use but frequently provides a much better transfection efficiency. Transfections with lipofectamine 2000 herein named l2k. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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.
Without changing anything to the transfection conditions you have already setteld, combimag a specific magnetic formulation can be added to complexes of lipofectamine 2000 to raise the efficiency. The intracellular trafficking mechanism of lipofectamine. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Using lipofectamine 2000 invitrogen the transfection efficiency was about 14 % and. Tm328 fugene hd transfection reagent technical manual. Cells in a 6 well dish, plated and at 70% confluence. 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. Comparison of transfection efficiency of nonviral gene. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Transfection protocol lipofectamine 3000 transfection reagent mcf10a.
What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. 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. What recommendations do you have for selecting a transfection reagent. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Cells in a 6 well dish, plated and at 90% confluence. Despite this, a satisfactory mechanismbased explanation of their. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Some of my cells will dried if i used the volume suggested by the manual. Comparative transfection of dna into primary and transformed. By the way, the protein size i tried to transfer is 65 k. Transfection protocol lipofectamine 3000 transfection.
Lipofectamine 3000 protocol written out with timing specifics. First lipoplexes are bound to the cell surface and show slow transport behavior. Lipofectamine 2000 dna transfection reagent protocol. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios. The buyer cannot sell or otherwise transfer a this product b its components or c.
Invitrogen lipofectamine 3000 transfection reagent from. Lipidbased transfection supportgetting started thermo fisher. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Invitrogen lipofectamine 2000 transfection reagent fisher. Luciferase expression was compared at 24 hours post transfection using a standard luciferase assay. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The highest transfection efficiency in many cell types and formats e. Add 6l of lipofectamine2000 from biostores and 100 l of. 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.
A sample protocol is listed here for transfection experiments performed in 6well plates. Mar 03, 2014 for transfection of cells only with optimem i reduced serum media, the lipofectamine 2000 transfection mixtures were prepared by addition of 2. Luciferase expression was compared at 24 hours post. For additional information on scaling your transfection reaction, see page 4.
Lipofectamine 2000 transfection protocol 10cm plates reagents. By contrast, active transport along microtubules results in dna degradation. The intracellular trafficking mechanism of lipofectaminebased. Higher densities can lead to spontaneous differentiation. Do any one has protocol for stable transfection with lipofectamine 2000. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. An electroporation protocol for efficient dna transfection in pc12. I am about to start my stable transfections with lipofectamine 2000. You can generally use other serumfree medium instead of optimem. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Lipofectamine plasmid transfection bridges lab protocols. I use 24well plates for transfections in triplicates.
I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Highest transfection efficiency in many cell types and formats e. Maintain p19 cells undifferentiated in mem with 10% serum 7. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent. The trusted, simple, broadspectrum transfection reagent for most cell lines. 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. For using lipofectamine 2000 reagent, the above plasmids were. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. An improved method for increasing the efficiency of gene. I want to try to use hela cell and lipofectamine 2000 to do. Rnai or technical service, as are celltype specific rnai transfection protocols see rnai protocols.