Lipofectamine 3000 transfection protocol

Lipofectamine is quite efficient, you can prepare reagent according to standard protocol, first replace complete media DMEM +10 FBS with OptiMEM than adding DNA lipofectamine complex into it with... Figure 1. Superior knockdown with Lipofectamine RNAiMAX Transfection Reagent compared to competing siRNA transfection reagents. At both 10 nM and 1 nM p53 siRNA, Lipofectamine RNAiMAX Transfection Reagent provides more effective knockdown relative to nontransfected cells than other RNAi reagents, including siLentFect™ (Bio-Rad), DharmaFECT™ (Dharmacon), and HiPerFect (Qiagen) reagents. Transfection starts 2-3 days after seeding the cells (70% confluency). I basically followed Lipofectamine 3000 protocol. 1 ug Plasmid and 0.75 uL Lipofectamine were provided to each well. 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: Highest transfection efficiency in many cell types and formats ( e.g. 96-well). TRANSIENT TRANSFECTION OF MAMMALIAN CELLS WITH LipofectAMINE. Lan Zhou 11 /01/00, Edited by TCH 1/21/02. General Note: Plate cells for 2-12hrs (but not over 24 hours) prior to transfection to achieve a 40-80% confluence at the time of transfection. Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Successfully transfected cell types include: View performance data for 60+ cell lines plus comparison to Lipofectamine 2000 > 2Optimum amount needed is determined from the protocol (see pages 2–3). Co-Transfection of Plasmid DNA and siRNA Transfect plasmid DNA and siRNA at the same time using Lipofectamine® 2000 Reagent by adding 30 pmol (~0.6 μg) of siRNA per 1 μg of DNA. mRNA Transfection mRNA can be transfected in a 24-well plate by using Lipofectamine® 2000 DNA transfection experiments For immunocytochemistry experiments DNA transfection was performed using Lipofectamine 3000 reagents, following the manufacturer’s protocol (Thermo Fisher Scientific). .. In order to normalize transfection efficiency, a fixed amount of DNA from pEGFP construct expressing enhanced green fluorescent protein (GFP ... ** Optimum amount needed is determined from the protocol for Lipofectamine 3000 Transfection Reagent. For your convenience, the essential components of this protocol are now available in the Gibco™Lung Cancer Starter Kit. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. Transfection using Lipofectamine 3000 was performed according to the manufacturer's protocol with a DNA to Lipofectamine ratio of 1 : 3 w/v. A transfection enhancer, the 3000 enhancer reagent (1 : 2, DNA : Reagent, w/v), was used along with the Lipofectamine 3000 transfection reagent for all transfections. Aug 15, 2015 · The protocol for Lipofectamine 3000 LV production requires high cell density, 95-99% confluence at the time of transfection. This prevents HEK293T (or FT) cells from detaching from the culture vessel and causing any interruption due to adding DNA/reagent complex, changing media, harvest LV by replacing fresh media. Feb 22, 2013 · Materials. Cells in a 6 well dish, plated and at ~70% confluence. Typically transfect as myocytes. Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL) Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. A low toxicity lipid dose (0.75µL for 24-well plates) is suggested for applications requiring minimal disruption of the cells. Learn more at http://www.lifetechnologies.com/transfection Optimized protocol for Lipofectamine LTX & Plus reagent: http://tools.invitrogen.com/content/sfs/m... This is a protocol I used for transfection of HeLa in 6 well plates (volumes per well): Mix 2.5 µg plasmid DNA with 5 µl P3000 in 125 µl OptiMEM. Then add 3.75 µl Lipofectamine 3000 in 125 µl... 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: Highest transfection efficiency in many cell types and formats ( e.g. 96-well). Learn more at http://www.lifetechnologies.com/transfection Optimized protocol for Lipofectamine LTX & Plus reagent: http://tools.invitrogen.com/content/sfs/m... Protocol Pub. No. MAN0007825 Rev.1.0 Lipofectamine® RNAiMAX Reagent Protocol Outline A. Plate cells so they will be 60-80% confluent at the time of transfection. B. Prepare RNA-lipid complexes. C. Add RNA-lipid complexes to cells. Lipofectamine® RNAiMAX Transfection Protocol See page 2 to view a typical RNAiMAX transfection procedure. I was using Lipofectamine® to transfect HEK cells and was only getting about 20% transfection rates but after using TransIT®-2020 it went from 20% to 70%! I highly recommend using this reagent. It was very easy to follow the protocol and I obtained high cell viability. (Review published in SelectScience) Lipofectamine® 3000 is a versatile reagent that can also be used to deliver siRNA using the same transfection protocol. Simply substitute siRNA for DNA. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using Lipofectamine ® 3000, Lipofectamine ® 2000, and Protocol Pub. No. MAN0007825 Rev.1.0 Lipofectamine® RNAiMAX Reagent Protocol Outline A. Plate cells so they will be 60-80% confluent at the time of transfection. B. Prepare RNA-lipid complexes. C. Add RNA-lipid complexes to cells. Lipofectamine® RNAiMAX Transfection Protocol See page 2 to view a typical RNAiMAX transfection procedure. DNA transfection experiments For immunocytochemistry experiments DNA transfection was performed using Lipofectamine 3000 reagents, following the manufacturer’s protocol (Thermo Fisher Scientific). .. In order to normalize transfection efficiency, a fixed amount of DNA from pEGFP construct expressing enhanced green fluorescent protein (GFP ... Jan 17, 2019 · Protocol Overview The kit can be used by following the manufacturer's protocol. Make sure your cells are in antibiotic-free media to ensure optimal transfection. Plamid DNA is diluted in OptiMEM (invitrogen) and P3000 reagent is added to the diluted at 1:2 ratio. Lipo3000 reagent is diluted in OptiMEM in a separate tube. Learn more at http://www.lifetechnologies.com/3000 A persistent challenge in genome editing is really obtaining a high transfection efficiency that is reprod... Lipofectamine is quite efficient, you can prepare reagent according to standard protocol, first replace complete media DMEM +10 FBS with OptiMEM than adding DNA lipofectamine complex into it with... IMLF can be transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) but transfection efficiency is higher using recombinant adenovirus (described later), which gives very good expression in a short-time relative to other methods. Figure 1. Superior knockdown with Lipofectamine RNAiMAX Transfection Reagent compared to competing siRNA transfection reagents. At both 10 nM and 1 nM p53 siRNA, Lipofectamine RNAiMAX Transfection Reagent provides more effective knockdown relative to nontransfected cells than other RNAi reagents, including siLentFect™ (Bio-Rad), DharmaFECT™ (Dharmacon), and HiPerFect (Qiagen) reagents. This is a protocol I used for transfection of HeLa in 6 well plates (volumes per well): Mix 2.5 µg plasmid DNA with 5 µl P3000 in 125 µl OptiMEM. Then add 3.75 µl Lipofectamine 3000 in 125 µl... Cas9 KO plasmid transfection of p47phox in sinus nodal cell was performed using a Lipofectamine 3000. Plated cells were exposed to the Cas9 KO plasmid or empty vector control. After this, Ang II (20 µM) was added to the culture medium for 5 days. IMLF can be transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) but transfection efficiency is higher using recombinant adenovirus (described later), which gives very good expression in a short-time relative to other methods. Lipofectamine 3000 reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. It delivers exceptional transfection efficiency into the widest range of difficult-to-transfect and common cell types (Figure 1) with improved cell viability. Figure 1. Protocol Pub. No. MAN0007825 Rev.1.0 Lipofectamine® RNAiMAX Reagent Protocol Outline A. Plate cells so they will be 60-80% confluent at the time of transfection. B. Prepare RNA-lipid complexes. C. Add RNA-lipid complexes to cells. Lipofectamine® RNAiMAX Transfection Protocol See page 2 to view a typical RNAiMAX transfection procedure. Lipofectamine ™ 3000 Reagent Protocol. For support visit thermofisher.com/support Transfect cells according to the following table. Use the indicated volume of DNA and . P3000 ™ Reagent with each of the two volumes of Lipofectamine 3000 (when performing optimization). Each reaction mix volume is for one well . and accounts for pipetting variations. I am currently having problems with my transfections and request help. I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% ...