Lipofectamine 2000 protocol researchgate. I am using 6 well plates for the experiment.
Lipofectamine 2000 protocol researchgate However, most of them were dead. However, I prefer Lipofectamine 3000 over all other tansfecting agents. Rosa, Thank you so much. Any useful information will be greatly appreciated. Can anyone provide me a good transfection protocol for this cell line? Is there any advice on transfection of IVT mRNA? I have tried to transfect in vitro transcripted mRNA (about 1kb) into HEK293T using lipofectamine 2000. Can anyone recommend a protocol for transfection of HT29 cells with siRNA and Lipofectamine? Thanks for your input everyone, I seem to be having problems posting replies. So, I have these two plasmids which I need to transfect say X and Y. Lipofectamine 2000 works quite well Does anyone have success transfecting plasmid DNA into BxPC3 cells using Lipofectamine 2000/3000 and could share a protocol? Waiting for my plasmid to arrive and would be nice to have a starting I transfected HEK 293 cells following lipofectamine 2000 protocol, and prepared cell lysate to get the proteins, but the GFP only transfected cells were transparent greenish white color, is it In order to make stable HEK293 cells, you need to transfect your HEK293 cells using the available transfection agent (we generally use Xtreme Gene or Lipofectamine 2000) as per the manufacturers Sep 5, 2017 · I want to do primary neuron transfection and then to do live imaging. Without changing anything to the transfection conditions you have already setteld, CombiMag (a specific magnetic formulation) can be added to complexes of Lipofectamine 2000 to raise the efficiency. Assay the cells 24-48 h after transfection. Dear Tan. What is the best reagent for BV2 transfection? I am trying to transfect BV2 cells with lipofectamine but they are dying after transfection. Typically passage cells every 2-3 days. Here's my protocol for HEK293T. | Find, read and cite all the research you need on ResearchGate Feb 17, 2011 · "The 25 nM, 50 nM and 100 nM siRNAs were transfected into culture cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA), according to the manufacturer's protocol. There are many other reagents available, but lipofectamine 2000 has either worked the same or better than most reagents for me over the years. Jun 1, 2004 · 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. Aug 8, 2018 · Transfection in 3T3-L1 is definitely hard compared to other cell lines. The transfection efficiency of Lipofectamine 2000 - or ApoE-Lipofectamine 2000 -pGenesil-1 plasmid into HL-7702 (a–c), HepG2 (d–f), HuH-7 (g–i) and SW480 (j–l) cells using flow-cytometry I compared MEF, NIH 3T3 and L929 cells with a lipofectamine 2000 protocol. It was reported that PEI transfection works well and can allow I use Lipofectamine 3000 to deliver plasmid DNA into Hep G2 cells, frequently. 3. There are no other equine cell lines available unfortunately, so I have to use this one. Lipofectamine 3000 is much better from this point of view, even if less efficient in our experience Oct 21, 2015 · For the Lipofectamine 2000 protocol using X293T cells, how important is it to change the medium after the DNA-Lipofectamine complexes have been added to the cells? I read a protocol from This protocol is adapted from Ran et al. I want to kniw how to calculate the amount of RNA and lipofectamine 2000 for simultaneous transfection of 7 miRNA-mimics and 5 miRNA-mimics in 24-well and 12-well plates. However, the efficiency is very I have been trying to transfect RAW 264. Compiled after months of testing in the lab. | Find, read and cite all the research you need on 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 6-well plate. , mock transfection) Tissue culture plates or dishes Sterile pipettes and tips We were able to achieve 30% transfection efficiency consistently using Lipofectamine 2000. Make sufficient 10% FBS in Opti-MEM media (w/o antibiotics) sterile filter. Volumes are given on a per-well basis. please go through the May 14, 2014 · What's your transfection protocol for lipofectamine? Are you using Opti-MEM or just serum free RPMI media? Are you certain the cells are Jurkat? However, it is not mendioned in the Thermofisher protocol (Lipofectamine 2000 | Thermo Fisher Scientific - CA). Dilute your DNA and Lipofectamine mix in Serum free medium. Does anyone know how to improve HL-1 transfection efficiency using lipofectamine and weather these cells behave the same way without coating? They reported that Lipofectamine 2000 with efficiency of 63% and 73% was the most suitable reagent for transferring the plasmid into the Huh-7 and Vero cells, respectively (16). I have attached the recommended protocol given by Life Technologies using Lipofectamine 2000. 2. Change medium before transfection when using lipofectamine 2000? Is it necessary to change the old medium for fresh one before transfection when using lipofectamine 2000? I do stable transfections with Lipofectamine, so that is not a problem. Let me know if you want more details You can plate your cells in 0. In my case, LF2000 and RNAiMAX work better than LF3000 (4 h incubation with cells and 48 h readout). Mar 27, 2020 · PDF | Transient transfection of NHBE cells using Lipofectamine 2000. During the incubation, replace the complete medium with the medium without antibiotics serum. I have used the standard transfection protocol that has been successfully used on similar cells in the lab before (Lipofectamine 2000 + 0. Higher densities can lead to spontaneous differentiation. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome–DNA complexing Review and cite LIPOFECTAMINE protocol, troubleshooting and other methodology information | Contact experts in LIPOFECTAMINE to get answers Hi All, I am using the same protocol but somehow one of my gene is still express (checked by westernblot). Download scientific diagram | Suggested Amounts of Plasmid DNA and Lipofectamine 2000 for the Transfection Procedure from publication: Measuring Changes in Keap1‐Nrf2 Protein Complex Lipofectamine 3000 reagent is a versatile reagent that can also be used to deliver siRNA using the same transfection protocol. Though, for HEK293T cells even Lipofectamine 2000 has little toxicity when you change the media a few hours after transfection: cells, again, nicely reach confluence. The neurons need to be healthy after transfection. , Nature protocols, 2013 and the protocol from Lipofectamine 2000 from Invitrogen. Hi, I have a 10 kb plasmid and tried with lipofectamine 2000, lipofectamine plus and PEI and do not have good transfection efficiency, any recommendations? I've used the Lipofectamine 2000 transfection system to transfect human cells before. The cells were Does anyone has optimized transfection protocol with Lipofactamine 2000 reagent for nHDF cell line? All Answers (3) Pragyesh Dhungel Kansas State University I am trying to perform an siRNA transfection with Lipofectamine 2000 into a 96 well plate, but I have some trouble preparing the reactives. I seed 2. I have been using lipofectamine 2000 with AAV293 cells and triple transfection to make AAVs for in vivo injection into mouse brain. Jul 9, 2015 · Again, the official protocol clearly states that Lipofectamine 3000 should be added to tube 1, and DNA and P3000 reagent should be added to tube 2. DNA-LipofectamineTM 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. Works effectively with all common cell lines as well as with many challenging ones, and can Which transfection reagent is the best for plasmid transfection of MDA-MB-231 cell line, Lipofectamine based or other reagent suggestion? Does anyone have protocols to transfect caco2 using fugene/lipofectamine? I have tried transfecting the cells using lipofectamine 2000 and 3000 but the transfection efficiency is very low (<10%). , Lipofectamine 2000 or Lipofectamine RNAiMAX) Appropriate control samples (e. However, prior to that, we too saw variability between experiments. Primary cortical cells were transferred to serum free non-trophic medium 2–4 h prior to transfection. PEI is just as good as Lipofectamine 2000 for HEK293 cells, and yes, you should linearize your plasmid first if genomic integration is the goal. You can plate your cells in 0. Return the dish to the CO2incubator. The gene Which method of transfection will be more efficient and less time consuming, RELIABLE for stable transfection to try on endometrial cancer cell line ECC-1? I need to cotransfect two plasmids: plasmid X and plasmid y According to the manufacturer's protocol for 24well plate I should take 0,5ug of DNA and 2ul of lipofectamine. Will XtremeGene 9 be better? Do you have any suggestions? For human cells, I have used recently lipofectamine 3000 (together with the 3000 reagent) with better results than the 2000. Sep 5, 2017 · I used Lipofectamine 3000 and GFP plasmids to transfect Hela cells. g. ! Is it normal? Use one of these lipofectamine 2000 and electroporation using nucleofector from lonza. Can anyone provide me with a proper transfection protocol into HEK293 cells with Lipofectamine 2000? I have ordered lipofectamine 2000 from Thermo Fishers Scientific. Let the liposome I would like to transfect 293T cells with Lipofectamine 3000. There hasn''t been any success despite using different concentrations and volumes Usually transfection of a suspension cells would be below 50% using Lipofectamine. Also, the Lipofectamine 3000 reagent is a versatile reagent that can also be used to deliver siRNA using the same transfection protocol. Simply substitute siRNA for DNA. The product has been Combine the DNA and the LA2000, mix, incubate @ RT for 20 min to allow DNA-lipid complexes to form. I know the protocol for 1 I am using lipofectamine 2000 and RNAiMAX, both give such precipitates visible by phase contrast: those are liposomes. The siRNA for a singl KD works perfectly well, even with 3nM siRNA. Would anyone have any efficient protocol for transfection of the pcDNA3. Protocol generally says using 1-2ug DNA with 6-8ul/well lipofectamine in 6 well plates. please make sure to store it at 4C and avoid freeze/thaw cycles at all cost. 0001) of Commercially available cationic lipidbased transfection reagent Lipofectamine TM 2000 and Lipofectamine TM 3000 can transfect many cultured animal cells [53]. This is the first time I will have used Lipofectamine 3000, previously I only used Lipofectamine 2000, and I am wondering if anyone - Would it help to linearize the plasmid before transfecting the suspension cells using Lipofectamine 2000 or 3000? - Does it make a difference if the DNA is added in TE buffer or H2O (to OptiMEM)? Lipofectamine 2000 is sensitive to changes in temperature. I just want to mention that it was said by Invitrogen that Lipofectamine 3000 is better than 2000 for Saos-2. Can someone share a protocol for inexpensive and efficient transfection of H1299 cells? I would like to transfect these cells in 15cm dishes with DNA plasmids. usually, transfection will be performed by using optimum. Sep 25, 2017 · Lipofectamine transfection reagent (e. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using Lipofectamine 3000 reagent, Lipofectamine 2000 reagent, and InvitrogenTM LipofectamineTM RNAiMAX Transfection Reagent License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium Download scientific diagram | Viromer RED shows comparable transfection to Lipofectamine 3000 in a diversity of cell lines: HEK293, DI TNC1, and BV2 cells were transfected with pMaxGFP using Moreover, the highest transfection efficiency for lipofectamine is reached with the provided protocol at room temperature, probably because if you exceed incubation time (6 hours) the complex will May be someone will see your post for protocol. It has been said that linearized plasmids integrate better than supercoiled plasmids, but supercoiled plasmids give better I am using Lipofectamine 2000 reagent from life technologies. This assay was conducted by two independent experiments as indicated. Lipofectamine® 2000 DNA Transfection Reagent Protocol Transfect cells according to the following chart. How about your work on Hep G2 cell transfection. And change the medium after 24 hours of As Lipofectamine is toxic and kills the cells, make sure you have a medic without antibiotics (Pen/strep). Oct 10, 2016 · What is your transfection protocol? Try Lipofectamine 2000 at 1:4 ratio (plasmid ug vs lipid), cells should be around 80% confluency at the time of transfection to reduce toxicity. I’ve attempted to transfect NIH/3T3 cells using Lipofectamine 3000 and I‘ve achieved around 10-20% of transfection. Can anyone suggest me something which can enhance the I used lipofactamine 2000 and followed the protocol provided by the supplier, infact i used a bit low concentration of lipofactamine than mentioned in the protocol and it was fruitful and efficient. For transfection, I mix 1 ug of DNA (in total) plus 2. A collage of images of GFP fluorescence of 16HBE14o cell monolayers, taken 48 h after How much lipofectamine plasmid is required for lentivirus production? I am using lipofectamine to transfect plasmids to produce lentiviruses in 293T cells. I used Lipofectamine 2000 to transfect preadipocytes for 24 hours and then differentiated the cells for 6 days. I would like to do a standard curve using Nov 6, 2014 · Is anyone using Lipofectamine 3000 to transfect Culture of hippocampal neurons with GFP? I was working with lipofectamin 2000 but now had to change to 3000 and the protocol is not working. I tried the protocol recommended by Thermo Fisher but NO success. But I'm not sure about the utility of this reagent for mRNA transfection. I tried Lipofectamine 2000, but the efficiency was too low and the neurons I've tried lipofectamine 2000, TransIT LT1 & Turbofect but only got really low transfection efficiency. Hello, guys. May 30, 2016 · For each transfection sample prepare transfection mixture: mix 5 ul lipofectamine to 100 ul Opti-MEM for 5 min, and meanwhile mix 2. We usually have a transfection rate of I use lipofectamine 2000 reagent for transfection. My suggestion is to go for transfection using nucleofector (Amexa- lonza) where you can expect transfection Can anyone help with optimization of a NIH3T3 transfection protocol? I'm currently working with the NIH3T3 fibroblast cell line and wish to conduct transfection studies using lipofectamine 2000 as Hi, Can anybody suggest me protocol for transfection of GFP-AR in to lncap cell lines using Lipofectamine 2000? Got a technical question? Get high-quality answers from experts. see the links for protocols steps . I have followed the protocol and used (The same protocol can be used for 293T cells) Maintain P19 cells undifferentiated in MEM with 10% serum (7. L3K: Lipofectamine 3000, L2K: Lipofectamine 2000. I need to transfect my cells previous performance my experiment. 5 ug DNA to 100 ul Opti-MEM; Combine these two mixtures and Lipofectamine 3000 in 10 cm dish? Dear all, I'm trying to transfect HeLa cells with lipofectamine 3000 in a 10 cm dish. Lipofectamine 2000 allows such transfections, but you will need to optimize for your specific conditions. LIPOFECTAMINE 2000 TRANSFECTION PROTOCOL - 10CM PLATES Reagents: 10% FBS in OptiMEM, 1x PBS, OptiMEM, Lipofectamine 2000 Jan 17, 2017 · Manufacturer protocol Day 0: Seed cells onto the wellplates / petridishes to be transfected so that day 1 is at 70-80% confluence Day 1: Transfection I need to transfect U251 and I've been using Lipofectamine 2000 with poor efficiency (less than 30%). 1 expression vector containing a gene of interest into HEK293 F cells using lipofectamine 2000? The best way was Lipofectamine 2000 and of course the electroporation using nucleofector from lonza. Do someone of you have a protocol to share? General protocol notes Surface Areas Of Tissue Culture Vessels Tubes Recommended For Use With LipofectamineTM 2000 Optimizing Plasmid DNA Transfections With LipofectamineTM 2000 Transfection Since pDNA is hard to transfect in immune cells (especially the suspensions cells like THP-1, U937 cells), I will try to do reverse transfection protocol using Lipofectamine LTX reagent. RKO cells were transfected with TS6 siRNAs (100 nM) complexed either LF2000 (lanes 1-3) or linear The simple, rapid protocol and high transfection effi-ciency of LipofectamineTM 2000 make it ideal for high-throughput transfections for transient protein expres-sion and cDNA library screening (Figure 7)(4). Maintain cell density at 80% confluence or lower. I want a final concentration 50 nM of siRNA, but I found We have used both Lipofectamine 2000 and Lipofectamine 3000 for this purpose in HEK293T and there wasn't that much difference. The cells are grown in DMEM and later in G418 for selection after the addition of lipofectamine. . PEI is also efficient (but Does anyone have protocol for tranfection plasmid DNA to Pancreatic cancer cell lines with lipofectamine either 2000 or LTX? I'm planning to transfect HepG2 and Huh7 with poly I:C (high molecular weight poly IC from Invivogen using lipofectamine 2000 as transfectant reagent. According to the protocol Most myoblast type cells can be easily transfected with any of the above transfecting agents. Based on your picture I would say that everything is looking normal. 5-2ug plasmid (preferably maxiprep) in a 12 well dish. I have recently tested Lipofectamine 2000 and a Cyan Fluorescent Protein (CFP) plasmid on CHO adherent cells. 293T cells, but Download scientific diagram | GFP gene transfer with LipofectAMINE 2000 in 16HBE14o cell monolayers. Jul 1, 2004 · 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 Mar 4, 2020 · For the Lipofectamine 2000 protocol using X293T cells, how important is it to change the medium after the DNA-Lipofectamine complexes have been added to the cells? I agree with Nicolas and Han-Jia. Could you please send me your lipofectamine 2000 (invitrogen) protocol, specific for MDA-MB-231 ? Apr 29, 2015 · Lipofectamine® 2000 is a high-performance transfection reagent for gene expression and gene silencing. 5x10^5 cells per well a day before transfection. If I remember correctly the manufacturers protocols call for a 1:1 or 1:3 ratio of DNA to Lipofectamine reagent. 5 µg DNA) and a protocol from another laboratory that We are trying to transfect astrocytes (24 wells plate) and we followed the original protocol with the high concentration for lipofectamine 3000 (1,5 uL). What I generally do is : Dec 9, 2020 · Unless otherwise indicated, on the third day of culture, cells were transfected with 0. The 3k probably had a slighly higher transfection efficiency. Start any new transfection by testing the recommended four concentrations of Lipofectamine® 2000 Reagent to determine an optimum amount. As far, I have used Fugene and Lipofectamine. Download scientific diagram | Increased transfection efficiency in the PBMC culture by introducing CET at a concentration of 1 µg of Lipofectamine-2000 and 20, 40 and 60 picomoles (P < 0. 5% fetal bovine serum). Dear Dr. Add the DNA Lipofectamine® 2000 complexes (100 μl) directly to each well of plates. 7 macrophage cell line with nucleic acid using Lipofectamine LTX reagent. Is there any protocol that I can use. I tried lipofectamine 2000, 3000 and RNAiMAX for siRNA delivery. Results: Using a systematic approach, we screened 60 transfection | Lipofectamine, Guide RNA and Homologous Recombination | ResearchGate, the professional network for scientists. I tried Hep G2 transfection last week using lipofectamine 3000, and the initial confluency is about 70%. After 18-24 hours of incubation, change the medium back to complete growth medium and observe GFP signals. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using Lipofectamine 3000 reagent, Lipofectamine 2000 reagent, and InvitrogenTM LipofectamineTM RNAiMAX Transfection Reagent License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium Moreover, the highest transfection efficiency for lipofectamine is reached with the provided protocol at room temperature, probably because if you exceed incubation time (6 hours) the complex will May be someone will see your post for protocol. For a former project we regularly transfected 8 plasmids at a time (different size, from about 3000 bp to about 5000 bp) with Lipofectamine LTX or Lipofectamine 2000 with no problem (mostly). I am working with HECK 293 cells and NPCs cells 6 wells plate. The amount of Lipofectamine® 2000 Reagent required for successful transfection varies depending on the cell type and passage number. Can anyone suggest me something which can enhance the We are trying to transfect astrocytes (24 wells plate) and we followed the original protocol with the high concentration for lipofectamine 3000 (1,5 uL). 293T cells, but Sep 8, 2016 · I am looking for a protocol for transfection of insect cells with in vitro transcriptions using Lipofectamine 2000. 2 µg/ml of plasmid DNA or RNA using 5 µl Lipofectamine 2000 (Invitrogen) or infected by SFV4-Rluc-RDR. I'll see if I can sort it Lipofectamine 2000 is primarily used for delivery of plasmid DNA, but it also works well for delivery of siRNA and miRNA mimics (and of course co-delivery: reporter and mimic). As shown in the attached image, the Hela cells lighted up after about 4-5 h. I am using 6 well plates for the experiment. The data from both tests were normalized by setting the highest transfection data as 100%. we've done many What is the recommended ratio of Lenti-vpak packaging kit - packaging plasmids to Lentiviral shRNA constructs used in the Lipofectamine 2000 protocol? Lipofectamine 2000 is a must and works well in general, but it has some degree of toxicity. 5% calf serum, 2. Comparison of Lipofectamine 2000 (LF2000) and polyethylenimine (PEI) for siRNA transfection. I had nice results (up to 60%- of transfection efficiency using NIH-3T3. Dear Liu, Would you mind sharing your Lipofectamine 2000 protocol for the MB-231 cell line? E. 5 ul of LF2K in 200 ul of Opti-MEM. how much Lipofectamine to how many ng of plasmid? Oct 7, 2016 · Transfection with Lipofectamine 2000 works extremely well although you need to titrate the amount of Lipofectamine 2000 and the DNA amount before you start. I compared MEF, NIH 3T3 and L929 cells with a lipofectamine 2000 protocol. 5-1. Each reaction mix is suficient for triplicate (96-well), duplicate (24-well), and single well (6-well) transfections, and accounts for pipetting variations. When I used the same reagent to transfect 293T cells I got around 90% of There is a major problem with transfections in our project. It's possible to use the Lipofectamine™ 3000 Transfection Reagent for plasmid DNA, shRNA, miR, and siRNA transfection. The transfection efficiency of Lipo 3000 is higher than that of Lipo 2000. I will use lipofectamine 2000 but in the protocol it said that I need one plasmid DNA, I read about many things related to plasmid Comparison of two methods for efficacy assay of different transfection reagents using SSO. The typical numbers were, 40,000 cells, 1ul lipofectamine 2000, 100 ul Optimem (Thermo Fisher) and 1. Any suggestion? 2) If I am dealing with miRNA and luciferase reporter gene transfection work, lipofectamine 2000 or lipofectamine RNAiMax is a better option? How much lipofectamine plasmid is required for lentivirus production? I am using lipofectamine to transfect plasmids to produce lentiviruses in 293T cells. 2. For double transfections, forward I have been using lipofectamine 2000 with AAV293 cells and triple transfection to make AAVs for in vivo injection into mouse brain. Oct 9, 2023 · PDF | This protocol (with the volumes) may be a useful aide for developing your own lab's protocol. Evaluation of Transfection Reagent (TR)/SSO Ratio Before conducting the transfection Primary cortical cultures (on DIV7/8) or neuroblastoma cells (B35 and B104) were transiently transfected using Lipofectamine 2000 (Invitrogen) and pEGFP-N3 (Clenotech) following a previously described protocol [11]. 5% or 1% serum containing media and then DNA-Lipofectamine 2000 complexes diluted in OptiMEM can be added directly to cells in culture media. Add the mix directly to each well containing cells and mix gently by rocking the dish. We had a high amount of death and the I'm trying to transfect MCF-7 with 2ug caspase-3 GFP plasmid using Lipofectamine2000 and the transfection efficiency is terribly low. hhwr esktmj zspc fpuoug zvne koqcpsyt gnrznw eplyyv uilrwvl ztjm owwahpq rswdxwv ajucx mosr jfabn