Invitrogen lipofectamine 2000 transfection reagent 0. For additional information on scaling your transfection reaction, see page 4. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. To make the fluorescence density of different treatment groups comparable, the manual mode was used to image fam labelled sso, with a fixed 4. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Add 6l of lipofectamine2000 from biostores and 100 l of. Do any one has protocol for stable transfection with lipofectamine 2000. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. 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.
Nov, 2002 lipofectamine 2000 reagent is a cationic lipidbased transfection reagent designed to give extremely high transfection efficiencies and result in equally high protein expression levels. Lipofectamine 2000 transfection reagent from invitrogen. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. This protocol is well adjusted for cancer cell lines. You can calculate this according to instructions in your reagent manual. Systematic screening of commonly used commercial transfection. Lipofectamine 2000 transfection reagent is a proprietary formulation.
Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem. Higher densities can lead to spontaneous differentiation. Maintain p19 cells undifferentiated in mem with 10% serum 7. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine transfection reagent is a value alternative for transfection of plasmid dna into eukaryotic cells with a simplified protocol. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. 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. Scaling up or down lipofectamine 3000 reagent transfections. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit.
We produce lenticdna viral stocks in 293t cells using the transfection conditions summarized in a table below. Lipofectamine 2000 transfection reagent dartmouth college. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. The amount of lipofectamine 3000 reagent for successful transfection varies. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Biomodule transfection and control user manual unit with.
So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for. It is possible to plate and transfect the cells on the same day, but. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection protocol lipofectamine 3000 transfection reagent. Start any new transfection by testing the recommended two concentrations of lipofectamine 3000 reagent to determine an optimum amount. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectaminez 2000 for transfection provides the following advantages. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. What percentage confluency should be used for stable transfection.
Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules, short interfering rna sirna or. Follow standard cloning protocol to pcr amplify the gene of interest goi and clone into the multiple cloning. Transfection with lipofectamin 2000 invitrogen culture vessel 96 well 48well 24well 12well 6well 35 mm 60 mm 100 mm 150 mm t25 t75 surface area cm2 0. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. The manufacturers of transfection reagents, the suppliers of lentiviral packaging constructs, and many academic laboratories have provided protocols for producing lentiviral stocks. Invitrogen lipofectamine 3000 transfection reagent l3000008. Lipofectamine 2000 dna transfection reagent protocol. Refer to the transfection collection and the invitrogen transfection guide available at for a list of cell lines and cell types that have been successfully transfected. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. I use 24well plates for transfections in triplicates. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Invitrogen lipofectamine 2000 transfection reagent life. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement contained in this user manual. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Use lowpassage cells, and make sure that cells are healthy and greater than 90% viable before transfection. I am about to start my stable transfections with lipofectamine 2000. It is very easy to use, somewhere between lipofectamine and fugene 6. Invitrogen transfection guide available at for a list of cell lines. Life technologies corporation andor its affiliates warrant their products as set. Lipofectamine rnaimax has a broad peak of activity. Invitrogen lipofectamine 3000 transfection reagent. The highest transfection efficiency in many cell types and formats e.
Highest transfection efficiency in cells at 90% confluency. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. The following procedure is provided as an example only. Lipofectamine 2000 reagent thermo fisher scientific. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Despite this, a satisfactory mechanismbased explanation of their. 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. Highest transfection efficiency in many cell types and formats e. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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.
Lipofectamine 2000 for transfection provides the following advantages. Lipofectamine 2000 and lipofectamine rnaimax transfection. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. The transfection efficacy of lipofectamine 2000 was compromised by its. So far, i have been very impressed by the performance of lipofectamine 2000. Laboratories and 5 ng of prltk using lipofectamine 2000.
Do not add antibiotics to media during transfection as this causes cell death. Lipidbased transfection reagents exhibit cryoinduced increase in. Invitrogen lipofectamine 2000 transfection reagent 1. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Max % of positive cells after transfection of a gfpplasmid 3. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Online resources visit our product page for additional information and protocols.
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. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. 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. The trusted, simple, broadspectrum transfection reagent for most cell lines. Invitrogen already markets several transfection reagents and so to produce another you have to expect improvements over their existing products, as well as over competing companys reagents. Enable high transfection efficiency in novel genome editing applications. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Increased transfection efficiency with the freezethawed reagent was also seen with. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. How does transfection with lipofectamine 2000 work. We call this approach reverse transfection and we have developed two methods to reverse transfect cells. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Invitrogen lipofectamine 2000 transfection reagent.
It has also been optimized to work for many mammalian cell lines. Lipofectamine plasmid transfection bridges lab protocols. The intracellular trafficking mechanism of lipofectamine. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Refer to the cell lines database for a list of cell types successfully. Invitrogen lipofectamine 3000 transfection reagent fisher.
The average lentiviral titers in our preparations are around 56x 10. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine transfection of c2c12 cells bridges lab protocols. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules, short interfering rna sirna or plasmid dna to mammalian cells for rnai analysis. Optimemin ul, equal for dna and lipofectamine lipofectamine in ul 25. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. Rnaimax transfection reagent 778030, invitrogen, waltham, ma. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul. Transfection protocol transfection components component cat. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. 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. Lipofectamine 2000 transfection protocol 10cm plates reagents.