Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Photograph of expected results dna lipofectamine scaling up or down. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. 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.
Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 2000 transfection reagent is a proprietary formulation for the. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. To this aim, we used complexes of lfn 2000 with cy3labeled plasmidic dna. Protocol for dna transfection and luciferase assay using. Optimized conditions for successful transfection of human.
Transfecting sirna using lipofectamine rnaimax pin. See page 2 to view a typical dna transfection procedure. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Refer to the cell lines database for a list of cell types successfully. 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. Advanced transfection with lipofectamine 2000 reagent. An external file that holds a picture, illustration, etc. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Original article a comparative study of transfection of.
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. Invitrogen transfection guide available at for a list of cell. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Lipofectamine 2000 transfection reagent invitrogen yumpu. How can i optimize the transfection efficiency using. The protocal is from thermo fisher scientific important guidelines for transfection 1. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
Optimizing transfection lipofectamine 2000 cd reagent. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. I use 24well plates for transfections in triplicates. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Protocol for lipofectamine transfection in nih3t3 cells. Lipofectamine 2000 d after 20 min incubation add the dnalipofectamine mix to the cells 1 ml 5 ml, the total medium will be 6 ml 4. After 16 h, the transfection mix was removed and 20. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Dilute your dna and lipofectamine mix in serum free medium. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 transfection reagent from thermo fisher. Invitrogen lipofectamine 3000 transfection reagent.
The intracellular trafficking mechanism of lipofectaminebased. Double transfection of keratinocytes with lipofectamine 2000 invitrogen equimpent. Lipofectamine 2000 invitrogen optimem media gibco 6well polylysine coated plates mini production 100 mm plates polylysine coated plates for small production 150 mm plates polylysine coated plates for large production retroviral vector plasmid media. Dna lipofectamine complexes must be made in serumfree medium such as optimem reduced serum medium and can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. A drug master file dmf has been submitted to the fda. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. L in optimem invitrogen and incubated for 15 min at room temperature.
Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Rna was mixed with lipofectamine rnaimax invitrogen in a ratio of 1. Protocol generally says using 12ug dna with 68ulwell lipofectamine in 6 well plates. An improved method for increasing the efficiency of gene. Life technologies corporation andor its affiliates warrant their products as set forth in the life. Fortyeight h after initial transfection, the supernatant with viral particles was filtered through a 0.
Lipofectamine 2000 reagent thermo fisher scientific. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Contact life technologies technical service or your.
Adjust the amounts of components according to your tissue culture format. L containing the lipofectamine 20 00 transfection reagent, 5 ng of the aireexpressing vector, 100 ng of the minppgl4 vector, and 0. The intracellular trafficking mechanism of lipofectamine. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For using lipofectamine 2000 reagent, the above plasmids were mixed with 30 ul lipofectamine 2000 in. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection protocol 10cm plates reagents. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Different types of transfection reagents can be used for transfection, optimize the amounts of dna and liposomes for each type.
Lipofectamine 2000 dna transfection reagent protocol. 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. Despite this, a satisfactory mechanismbased explanation of their. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest. Combine diluted dna and lipofectamine, mix and incubate at room temperature for 20 min. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. The most efficient reagents were the cationic lipid reagents, lipofectamine 2000 and ltx, from invitrogen, which were able to genemodify up to 38% of huvec. Use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see scaling up or down. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold.
Lipofectamine 2000 from invitrogen biocompare product. Scaling up or down transfections to transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and medium used in proportion. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The trusted, simple, broadspectrum transfection reagent for most cell lines. Lipofectamine 2000 transfection reagent is a proprietary formulation. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Mscs transfected by lipofectaminetm 2000 invitrogen, grand island, ny, usa were used as controls. Lipofectamintm 2000 invitrogen, dnaplasmid of interest, medium full medium, reduced medium, minimal medium, 24well plate procedure.
Optimizing transfection of primary human umbilical vein. Contact life technologies technical service or your local sales representative for permission to crossreference the dmf. Fibroblast 3t3, cos7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 and lipofectamine rnaimax transfection. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. The mtt cell proliferation assay was performed using kit from fluka, ltd.
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. Hek293t cells were triple transfected using lipofectamine 2000 with the transfer vector e. Invitrogen lipofectamine 2000 transfection reagent 15ml. Highest transfection efficiency in many cell types and formats e.
Product is guaranteed for 6 months from the date of shipment if stored properly. Invitrogen lipofectamine 2000 transfection reagent. You can search for similar products in our product directory. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells.
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