293 transfection protocol lipofectamine 2000 pdf

The following morning, carefully aspirate the media. Hek 293 cells are efficiently transfected with lipofectamine. Invitrogen lipofectamine 3000 transfection reagent. What percentage confluency should be used for stable transfection.

The transpass cos293 transfection reagent is transpass d2 transfection reagent, a nonlipid cationic polymer, tested for the transfection of plasmid dna into cos and hek293 cell lines in a serumcompatible protocol. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation. The image below figure 1 is an example of the microarrays that can be created with this approach. For using lipofectamine 2000 reagent, the above plasmids were mixed. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine plasmid transfection bridges lab protocols. Transfection and genome engineering fisher scientific. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine transfection of c2c12 cells bridges lab. Do any one has protocol for stable transfection with lipofectamine 2000. Lipofectamine 2000 reagent thermo fisher scientific. We use it regularly for a range of cell lines such as hek293, cho and cos7 and obtain consistently greater than 90% efficiency. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Complex cotransfections of many plasmids can be efficiently transfected using the transpass cos293 transfection reagent.

Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. Lipofectamine 3000 protocol written out with timing. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Pdf transfection is the most popular experiments among biological scientist to study the gene expression. Highest transfection efficiency in many cell types and formats e. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of.

Cells in a 6 well dish, plated and at 90% confluence. Trojanporter was used as described in the protocol for a 6well plate. Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein compared to the. A guide to transient expression of membrane proteins in.

Can anyone provide me with a proper transfection protocol. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Also sometimes it helps to remaxi your plasmid, even though my dna quality. Add the transfection mix dropwise being careful not to dislodge the cells. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. 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. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. For consistent animal originfree transfection, use optipro sfm cat. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. I use 24well plates for transfections in triplicates. A sample protocol is listed here for transfection experiments performed in 6well plates.

To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Genetic transfection an overview sciencedirect topics. All transfections were performed in serumfree media for four hours followed by complete media supplementation. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. All products were used according to manufacturers protocol. A guide to transient expression of membrane proteins in hek293 cells for functional characterization. An improved method for increasing the efficiency of gene. Invitrogen lipofectamine 2000 transfection reagent. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Hek293 cells were transiently transfected with lentivirus expression.

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 total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. To transfect cells with sirna, follow the protocol as. Cd 293, 293 sfm ii, and vpsfm are media formulations known to inhibit. Scale your transfections up or down by proportionately adjusting the. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. For me, fugene and lipofectamine 3000 not 2000 worked the best, while the worst for me were pei and metafectene you should really try to optimize your protocol, it can increase the transfection efficiency by up to 40% in some cases.

The 293t cells come off the dish very easily, even with pipeting. Each tube gets 8mls of optimem, 32ug of gag pol, 12. If you can see successful transfection through a visual diagnostic e. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Incubate the cells for 18 h, or until the following morning. Carefully transfer the transfection mix to the lentix 293t packaging cells. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules. Original article an improved method for increasing the efficiency of. I have ordered lipofectamine 2000 from thermo fishers scientific.

Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Optimized conditions for successful transfection of human. Hek293 standard transfection protocol 24well plate. Transfection of hek293 cells using three different reagents trojanporter lipofectamine 2000 nanofectamine summary protocol the cells were plated the day prior to transfection. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market.

Transfection protocols thermo fisher scientific in. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. This protocol is for transfecting plasmid dna into mammalian cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Each reagent was used to transfect hek 293, hela, lncap, a549, and.

Higher densities can lead to spontaneous differentiation. Expifectamine 293 transfection kit protocol see page 7 to view a typical transfection procedure. Maintain p19 cells undifferentiated in mem with 10% serum 7. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Cellspecific transfection protocols thermo fisher scientific uk. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Plate cells so they will be 7090% confluent at the time of transfection.

Superior performance for cotransfection of sirna and plasmid dna. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Lipofectamine 2000 transfection reagent life technologies, cat. When washing monolayers or changing the media, pipette gently against the wall of the. The standard transfection protocol while resulting in better transfection. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. We did not find substantial transfection efficiency variations in. For additional information on scaling your transfection reaction, see page 4. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11.

Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Cells in a 6 well dish, plated and at 70% confluence. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Cd 293, 293 sfm ii, cd hybridoma may inhibit cationic lipidmediated transfection. Can anyone provide me with a proper transfection protocol into hek293 cells with lipofectamine 2000. Product isguaranteed for 6 months from the date of shipment if stored properly. Freestyle 293 expression system for largescale transfection of suspension 293 cells in a defined, serumfree medium freestyle max reagent human mesenchymal stem cells lipofectamine 2000 lipofectamine 2000 lipofectamine reagent for the transfection of dna into eukaryotic cells. Invitrogen lipofectamine transfection reagent protocols have been. Transfection conditions for expi293f cells for each 30ml transfection, use 7. We call this approach reverse transfection and we have developed two methods to reverse transfect cells. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression.