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Cells were transfected with plasmids containing either empty vector or ZO-1-3′UTR, using Lipofectamine 3000 (Invitrogen; catalog number: L3000-008). The cells were then transfected a second time after 36 hours with either miR-501-mimic or scr-miR (Applied Biosystems) and incubated for 24 hours. PEI transfection protocol 24-well protocol 1. Preparation of the transfection solution: For one well of a 24 well plate: 100,000 cells, 2µg of DNA and 10µl of PEI (25KD,

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Invitrogen™Lipofectamine™3000 Transfection Reagent is a highly effi cient, cost-effective tool for lentiviral production (Figure 1). This versatile reagent enables high viral titers even with genes that are large or diffi cult to package.Mar 24, 2020 · Briefly, 293T cells were transfected with pcDNA3.1.S2 (30 μg for a T75 flask) using Lipofectamine 3000 (Invitrogen, L3000015) following the manufacturer’s instruction. Twenty-four hours later, the transfected cells were infected with G*ΔG-VSV with a multiplicity of four. reddit

May 17, 2020 · At that time cells were either electroporated using the mNeon system (Invitrogen) using optimized settings for K562 cells, or lipofectamine 3000 again following manufactures instructions. Cells were then returned to 24 well plates with fresh media and grown for at least two days before being used in downstream analysis. Apr 29, 2020 · Briefly, the miR-21-targeting R-HCR mixture containing H 5 (0.05 nmol) and H 1 +H 2 +H 3 +H 4 +S+L (0.1 nmol each) in Opti-MEM (200 μl) was mixed with lipofectamine 3000 (5 μl) in Opti-MEM (200 μl) for 5 min. Then the above Opti-MEM transfection mixture and a supplementary 80 μl of FBS were introduced into the plated cells for 4 h at 37°C.

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Jun 14, 2017 · The erythrocyte membrane can be used as a cloak containing >3,000 nanosponges. Once they are fully loaded with toxins, nanosponges are safely disposed of by the liver with low toxicity. Therefore, nanosponges are designed to work with any type of cancer or poisoning that exhibits dysregulation of, or abnormalities in, cellular membranes. 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 (0.75 µL for 24-well plates) is suggested for applications requiring minimal disruption of the cells. Enable High Transfection Efficiency in Novel Genome Editing ApplicationsI use 2.5ul of Lipofectamine 3000 for each 1ug of plasmid each in 50ul optiMEM incubate for 20 min at room temp, then add to 1^6 of HEK293cells that plated in DMEM supplemented with 10% FCS, 48...

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Transfection optimization using the ViaFect™ Transfection Reagent. TF-1 cells were plated in growth media without antibiotics at 30,000 cells per well in a white 96-well assay plate and transfected with a CMV- luc2 plasmid using various lipid (ViaFect™ Transfection Reagent):DNA ratios.

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Life technologies lipofectamine 2000

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I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% confluency. Also, I used 2500 ng of DNA per well, 5µl of P3000 per DNA, 5µl ... Oct 11, 2015 · 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.

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Transfection optimization using the ViaFect™ Transfection Reagent. TF-1 cells were plated in growth media without antibiotics at 30,000 cells per well in a white 96-well assay plate and transfected with a CMV- luc2 plasmid using various lipid (ViaFect™ Transfection Reagent):DNA ratios. Feb 26, 2020 · The next day, f1 G8P PD plasmid (1.5 μg) were transfected into cells using Lipofectamine 3000 (Invitrogen). At 24 h post G8P PD transfection, plasmids encoding SaCas9 (0.75 μg) and EMX1 -targeting sgRNA or FANCY -targeting sgRNA (0.375 μg) were co-transfected into G8P PD -expressing HEK293T cells.

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Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells. 2.1.1. Optimization of SSLNPs for siRNA Delivery . Lipid-Z (detail on the lipid synthesis is provided under experimental section) was prepared, purified and characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) ().

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To generate virus, HEK293T, 293LTV or 293FT cells were transfected with lentiviral plasmids and packaging plasmids using Lipofectamine 3000 (Invitrogen); on the following day, the medium was replaced with fresh growth medium, and lentivirus-containing supernatants were harvested and concentrated by centrifugation. SIGMA Optimization Pro is the dedicated software that enables you to connect lenses from new product lines to your computer via SIGMA USB Dock, and to customize the lens with operations such as firmware update and focus adjustment.

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Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). The plasmid psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260) and pCMV-VSV-G was a gift from Bob Weinberg (Addgene plasmid # 8454) [15]. After 48 h, supernatant was harvested, filtered and added to Nalm6 cells with 4 μg/mL polybrene. All the transduced of Lipofectamine RNAiMAX can be obtained from the Invitrogen Website.) However, for cotransfection of siRNA/chimera and plasmid DNA, Lipofectamine 2000 should be used. To achieve the maximal RNAi activity and lowest cytotoxicity, optimization of Lipofectamine 2000 volume is essential. Aug 18, 2010 · Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to ...

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Jun 02, 2016 · Lipofectamine: Life Technologies: L3000075: Lipofectamine 2000, Lipofectamine 3000: Opti-MEM I: Life Technologies: 31985062: Bac-to-Bac Baculovirus Expression System: Life Technologies: 10359-016: Cimarec I 6 multipoint stirrer plate: ThermoFisher Scientific: 50094596: Polyclear ultracentrifuge tubes: Seton: 7025: Confirm appropriate tubes for ...

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Lipofectamine 3000 reagent transfection efficiency Protein expression improvement (x-fold), Lipofectamine 3000 vs. Lipofectamine 2000 reagents 3T3 4 4T1 2 A431 2 A549 3 ACHN 2 bEnd.3 9 BJ 3 BT-549 4 C2C12 3 C6 5 Caco-2 2 Caki-1 4 CHO-K1 1 CHO-S 1 COLO 205 4 COS-7 4 DU 145 2 H460 3 H9c2 3 HCC1937 5 HCT116 1 Cell type Lipofectamine 3000 reagent ...

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Oct 05, 2015 · Formulation of Lipofectamine 2000 and Lipofectin. LIPOFECTIN is a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and ... Lipofectamine®3000, a new transfection reagent developed to improve delivery and enable use of new technologies, can be used in more relevant systems enabling faster and more reliable outcomes.