To investigate the influence of medium and transfection time, primary hepatocytes were transfected with metafecte pro, lipofectamine 2000 or targefect in the presence of dmem figure 2a. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Procedural guidelines culture cells in serumfree media that are free of animalderived components. See page 2 to view a typical dna transfection procedure. Troubleshooting for transfection of the crisprcas9 using.
I am about to start my stable transfections with lipofectamine 2000. You can find in the instruction manuals of these two reagents a simple way of. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. After 4h aspirate media and refeed cells with normal media. Optimisation of dna transfection of the c2c12 muscle myoblast cell line, comparing use of polyethylenimine pei and lipofectamine2000. Optimising c2c12 muscle myoblast transfection with. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Our study demonstrated that a small selection of commercially available chemical transfection reagents was able to transfer exogenous genes efficiently to primary human cells. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Invitrogen lipofectamine 3000 transfection reagent.
The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions. Subjectivity of forces associated with manualmuscle test grades of 3. Plate cells so they will be 7090% confluent at the time 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. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. Invitrogen lipofectamine 2000 transfection reagent 15ml. Fugener hd transfection reagent technical manual tm328. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Lipofectamine plasmid transfection bridges lab protocols. Optimizing transfection of primary human umbilical vein. Test serumfree media for compatibility with lipofectamine 2000 cd reagent since some serumfree formulations e. Lipofectamine 2000 transfection reagent from thermo fisher. If there appears to be largescale loss of cells after lipofectamine. Cells in a 6 well dish, plated and at 70% confluence. Stealthsirna transfection protocol lipofectamine 2000. Lipofectamine 3000 reagent thermo fisher scientific tr. How does transfection with lipofectamine 2000 work.
Lipofectamine 2000 transfection reagent is recommended if you are transfecting a broad range of cell lines or want to transfect rna. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. 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. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Lipofectamine 2000 reagent thermo fisher scientific. The lenti293t cell line has been tested as a suitable cell line for lentiviral production.
Cotransfection of two plasmids tissue and cell culture. Advanced transfection with lipofectamine 2000 reagent. Cells should be plated on collagencoated dishes, as for normal propagation. Lipofectamine is formulated to give high transfection efficiency with little. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. But muscle, for example, will be hard to transfect. Transfection was performed as described in protocol 2 with the following. Invitrogen lipofectamine transfection reagent protocols have been. Comparative nucleic acid transfection efficacy in primary. Lipofectamine 2000 cd reagent 12566014 1 ml 4c 1 do not freeze. Transient transfection of mammalian cells with lipofectamine.
Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Comparison of three different transfection methods biology. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different. I have used both lipofectamine 2000 and 3000 and always get the.
The downsides include low efficiency, timeprohibitive testing necessary before doing largerscale transfections, and. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. 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.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Max % of positive cells after transfection of a gfpplasmid 3. The intracellular trafficking mechanism of lipofectamine. It is used to increase the transfection efficiency of rna. Ht76, human grade iii transitional bladder cell carcinoma, sirna, lipofectamine rnaimax.
Combine the two volumes of optimemdna and optimemlipofectamine. Despite this, a satisfactory mechanismbased explanation of their. Highest transfection efficiency in many cell types and formats e. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Lipofectamine 2000 dna transfection reagent protocol. 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. Plate cells for 212hrs but not over 24 hours prior to transfection to. Does lipofectamine 2000 give a stable transfection. Transfection protocol of lipofectamine rnaimax can be. Applied kinesiologists use submaximal manual break tests and a binary grading scale to test shortterm changes in muscle function in response to challenges.
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 of nih3t3 cells, hela, swis 3t3, 293t with. Both lipofectamine 2000 and 3000 work well for 293t cells, you can just. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Reverse transfection of stealth rnai or sirna using. The most commonly accepted method of evaluating muscle strength is the oxford scale aka medical research council manual muscle testing scale. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount.
Scaling up or down lipofectamine 3000 reagent transfections. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. Lipofectamine 2000 transfection reagent dartmouth college. General procedure for transfection of mammalian cells with. Lipofectamine transfection of c2c12 cells bridges lab.
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