It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. The fugene hd protocol database provides optimization guidelines for a. In another hand, i have tried the same lipofectamine under the given experiemental conditions to ht29 cells. To transfect cells with sirna, follow the protocol as described for dna but. Protocol for ht29 sirna transfection using lipofectamine. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Add 6l of lipofectamine2000 from biostores and 100 l of.
This reference provides a recommended procedure to transfect plasmid dna into ht29, human colon adenocarcinoma cells atcc no. If there is anyone who have tried best protocol to trasfect ht29 bothe reagent and condition, it will be really great to know. Lipofectamine 2000 dna transfection reagent protocol. Transfection protocol lipofectamine 3000 transfection. This protocol presents an efficient and reliable method to transfect human thp1 macrophages with sirna or plasmid dna by electroporation with high transfection efficiency while. It is best to use polypropylene instead of polystyrene tubes. The transfection efficiencies of the pte cells were 5% effectene. Protocol for ht29 sirna transfection using lipofectamine 2000. Transfection protocol lipofectamine 3000 transfection reagent ncih292 lung cancer cells complete growth medium component cat. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. Using lipofectaminez 2000 for transfection provides the following advantages. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000.
Advanced transfection with lipofectamine 2000 reagent. In a majority of cases, one wants the highest level of transfection. Aug 17, 2017 the pcmvmyc3mdm2 plasmid or small interfering rna was transfected into cells using oligofectamine invitrogen, 12252011 or lipofectamine 2000 invitrogen, 11668019 according to the. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. Tib202 is a human acute monocytic leukemia cell line that was derived from peripheral blood. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Invitrogen lipofectamine 3000 transfection reagent l3000008. Lipofectamine messengermax and the leading mrna delivery reagent were used to deliver gfp mrna 500 ngwell in a 24well format. Transfecting plasmid dna into ht29 cells using lipofectamine. However, significant toxicity from lipofectamine 2000 was also. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Comparison of transfection efficiency of nonviral gene. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection protocol lipofectamine 3000 transfection reagent. Enable high transfection efficiency in novel genome editing applications. I have got transfection efficiency of 3040% in sw620 and less than 30% in ht29. Transfection efficiency of p19 cells is between 3050%. Highest transfection efficiency in many cell types and formats e. In my experience lipofectamine works like a charm in many cells, including hek293. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%. I agree with parvoman that one of the main considerations is how well the cells are growing.
Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection guide overview of transfection methods promega. Transfecting plasmid dna into nih3t3 cells using lipofectamine ltx reagent. Transfection of ht29 cells with sirna and lipofectamine 2000 can anyone recommend a protocol for transfection of ht29 cells with sirna and lipofectamine. 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. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity.
Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. While lipofectamine 2000 reagent demonstrated transfection efficiencies. 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. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Ht29 transfection kit colorectal adenocarcinoma altogen. Lipofectamine is formulated to give high transfection efficiency with little. Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Genexplus transfection of plasmid dna into thp1cells.
I want to try to use hela cell and lipofectamine 2000 to do. Lipofectamine 2000 transfection reagent from invitrogen. Mdm2 promotes epithelialmesenchymal transition and. The mechanism of lipofectamine 2000 mediated transmembrane. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Explore our xtremegene transfection reagent recommendations guide for the.
Comparative transfection of dna into primary and transformed. Transfecting plasmid dna into nih3t3 cells using lipofectamine. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. This reference provides a recommended procedure to. Transient plasmid dna transfection into hela and hacat cells. Optimized conditions for successful transfection of human. Transfection was measured by luciferase activity using a conventional assay.
Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Using four different transfection reagents fugene hd, lipofectamine 2000, effectene, and lipofectamine ltx with plus reagent, jacobsen et al. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Dna complexes were added to cells that had already been allowed to.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. To perform transfection experiments in other cell culture plates, simply multiply the. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Xtremegene sirna transfection reagent polymer reagent for. Xtremegene hp reagent xhp, and competitor reagents ltx and l2k. 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. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Improved delivery of cas9 proteingrna complexes using. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. The vector contained an ofp reporter gene and was transfected.
Lipofectamine 2000 reagent thermo fisher scientific. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. I want to try to use hela cell and lipofectamine 2000 to. I am doing crisprcas9 transfection in sw620 and ht29 cell lines using lipo2000. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Invitrogen lipofectamine 3000 transfection reagent 0. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. It is used to increase the transfection efficiency of rna.
Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogen lipofectamine 3000 transfection reagent. Invitrogen lipofectamine 2000 transfection reagent fisher. A highly efficient method of transfecting hep g2 cells. Transient plasmid dna transfection protocol for ht29 cells per well of a 96well plate using xtremegene hp dna transfection reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For additional information on scaling your transfection reaction, see page 4. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Genscript recommends using lipofectamine 2000 for all transfections. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Transfection protocol of lipofectamine rnaimax can be.
A sample protocol is listed here for transfection experiments performed in 6well plates. Highly efficient transfection of human thp1 macrophages. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei. The transfection efficiencies of the pte cells were 5%. The mixture is used to transfect hek293 cells in a. Comparison of transfection efficiency of nonviral gene transfer reagents. Invitrogen lipofectamine 3000 transfection reagent fisher. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. I use 24well plates for transfections in triplicates. Dna complexes were added to cells that had already been allowed to attach to the plate b.
The pcmvmyc3mdm2 plasmid or small interfering rna was transfected into cells using oligofectamine invitrogen, 12252011 or lipofectamine 2000 invitrogen, 11668019 according to. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection of ht29 cells with sirna and lipofectamine 2000. Can anybody recommend a method transfection substrate of how to transfect ht29 cells. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Cells were transfected with a firefly luciferase encoding plasmid dna 0. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. This reference provides a recommended procedure to transfect plasmid dna into thp1 human monocytic leukemia cells atcc cat. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. 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. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Lipofectamine 2000 transfection reagent dartmouth college. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from.
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. Comparative transfection efficiency of jetprime versus its main competitor. Lipofectamine messengermax outperforms leading dna delivery reagent and leading mrna delivery reagent in various cell lines that are very difficult to transfect bend. Cells in a 6 well dish, plated and at 70% confluence. Choosing the right transfection reagent for optimal efficiency. 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. Xtremegene sirna transfection reagent polymer reagent. Transient plasmid dna transfection into hela and hacat. Refer to the cell lines database for a list of cell types successfully. Fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Invitrogen has taken the time to optimize this reagent and the recommend protocol that they provide. Help on transfecting mcf7 and hct 116 cells transfection.
In biontex experiments gfp positivity increased to 90. Lipofectamine transfection of c2c12 cells bridges lab. Xtremegene sirna transfection reagent is a proprietary blend of lipids and other components, free of animal products. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. In tube a place 2 g of dna in optimemi for a final volume of 200 l.