Genscript recommends using lipofectamine 2000 for all transfections. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 2000 transfection protocol 10cm plates reagents. Mean transfection efficiency with metafectene declined to 0. What is the major character of lipofectamine 2000 as a. Do any one has protocol for stable transfection with lipofectamine 2000. Out of the five transfection systems tested, fugene and rnaimax.
The transfection efficiencies of the pte cells were 5% effectene. Advanced transfection with lipofectamine 2000 reagent. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Original article an improved method for increasing the efficiency of. Transfection was measured by luciferase activity using a conventional assay. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Calibrate the system using a test plasmid with reporter gene function. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Highest transfection efficiency in many cell types and formats e. Invitrogen lipofectamine transfection reagent protocols have been. Prewarm 50ml of optimum stored in cold room at 4c 10min. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent.
Scaling up or down lipofectamine 3000 reagent transfections. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. In the liposome transfection, the quantity of lipofectamine 2000 could be reduced even further and this may produce a higher level of expression as lipofectamine can be toxic to cells. After 4h aspirate media and refeed cells with normal media. Transfection guide overview of transfection methods promega. A gfp green fluorescent protein plasmid can be used to determine transfection efficiency. This protocol is for transfecting plasmid dna into mammalian cells. Metafectene is superior to lipofectamine in the transfection of gs alpha prostate cancer cells.
How does transfection with lipofectamine 2000 work. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. Lipidbased transfection reagents exhibit cryoinduced increase in. Applied science or lipofectamine 2000 transfection reagent invitrogen. Start any new transfection by testing the recommended four concentrations of lipofectamine2000 reagent to determine an optimum amount. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. The reagent provides high transfection efficiency in many cells types and formats. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. General procedure for transfection of mammalian cells with. Start any new transfection by testing the recommended four. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. I use 24well plates for transfections in triplicates. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Enter your email address below and we will send you the reset instructions. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells.
Lipofectamine 2000 reagent thermo fisher scientific. To test this hypothesis, we evaluated the te of lfn and dd on both. Invitrogen lipofectamine 3000 transfection reagent from. Transient transfection of mammalian cells with lipofectamine. The intracellular trafficking mechanism of lipofectamine. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0. Among them, although rnaimax displayed the second highest transfection efficacy, and it had the best safety profile, with 83. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Cellspecific transfection protocols thermo fisher scientific us.
Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Invitrogen lipofectamine 2000 transfection reagent 15ml. An improved method for increasing the efficiency of gene. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Graphpad prism software and students ttest was used to. I want to try to use hela cell and lipofectamine 2000 to. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Maintain p19 cells undifferentiated in mem with 10% serum 7. Test serumfree media formulations for compatibility with lipofectaminez 2000 as some serumfree formulations can inhibit cationic lipidmediated transfection. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations.
I have used both lipofectamine 2000 and 3000 and always get the same results. Test serumfree media formulations for compatibility with lipofectaminez 2000 as some serumfree formulations can. Lipofectamine 2000 transfection reagent dartmouth college. Aug 07, 2012 the day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. Combine the two volumes of optimemdna and optimemlipofectamine. 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 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. 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. Protocol and application notes back to table of content. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. The same can be said for the calcium phosphate transfection. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine transfection of c2c12 cells bridges lab. Despite this, a satisfactory mechanismbased explanation of their.
Metafectene is superior to lipofectamine in the transfection. Cells in a 6 well dish, plated and at 70% confluence. Lipofectamine 2000 transfection reagent from thermo fisher. B hela cells were transfected with luciferase encoding plasmid dna using either transit. 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. Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine 2000 is an easy to use transfection reagent. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. The mechanism of lipofectamine 2000 mediated transmembrane. May 11, 2016 it is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8.
The media needs to be changed at least 1hr prior to transfection. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Systematic screening of commonly used commercial transfection. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. What percentage confluency should be used for stable transfection. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. Lipofectamine plasmid transfection bridges lab protocols. 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. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Troubleshooting for transfection of the crisprcas9 using. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection.
Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The technique requires optimization of pulse duration and strength for each type of cell used. For additional information on scaling your transfection reaction, see page 4. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine 3000, rnaimax, fugene and lipofectamine 2000 all displayed high transfection efficacy 97. Add 6l of lipofectamine2000 from biostores and 100 l of. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Max % of positive cells after transfection of a gfpplasmid 3. Comparative transfection of dna into primary and transformed.
Highest transfection efficiency in many cell types and formats. Dna complexes were added to cells that had already been allowed to attach to the plate b. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%. Luciferase activity was measured after 24 hours figures 24. With gs alpha cells, metafectene transfection of gfp plasmid alone yielded 46. A sample protocol is listed here for transfection experiments performed in 6well plates. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. It is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. Comparison of three different transfection methods biology.
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. Transfection is used to introduce a gene of interest into a cell. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.