Dharmafect 1

Manufactured by Horizon Discovery

Sourced in United States, United Kingdom, China

DharmaFECT 1 is a transfection reagent designed for the delivery of small interfering RNA (siRNA) and small hairpin RNA (shRNA) into mammalian cells. It is a cationic lipid-based formulation that facilitates the uptake of nucleic acids into the target cells.

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DharmaFECT 1 transfection reagent (405 citations) DharmaFECT (181 citations) DharmaFECT 1 reagent (121 citations) Transfection DharmaFECT 1 (3 citations) DharmaFECT 1 siRNA Transfection Reagents (2 citations) DharmaFECT 1 small interfering RNA (siRNA) Transfection Reagent (2 citations) DharmaFECT 1 lipid transfection reagent (2 citations) Dharmafect no. 1 reagent (2 citations) Dharmafect 1 (DF1; (2 citations)

520 protocols using dharmafect 1

siRNA Knockdown of Cell Cycle Regulators

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For siRNA treatment, Dharmafect 1 (Dharmacon, Lafayette, CO) was mixed in mTeSR1 with the appropriate siRNA according to the manufacturer’s instructions, then diluted with mTeSR1 and added to cells after aspirating old media. The final siRNA concentrations were: 100 nM siControl (Luciferase), 25 or 100 nM siCdt1, or a mixture of two siCdc6 (2144 and 2534 at 50 nM each). The Cdt1 siRNA mix was incubated on cells for either 20 or 24 hr, then changed to new mTeSR1 without siRNA. The Cdc6 siRNA mix was incubated on cells for 24 hr, then changed to new mTeSR1 without siRNA for 8 hr (32 total hours). The Cdt1, Cdc6 and Luciferase siRNA were described previously (Coleman et al., 2015 (link); Nevis et al., 2009 (link)). For siRNA treatment of RPE cells, Dharmafect 1 (Dharmacon) was mixed in Optimem (Gibco) with the appropriate siRNA according to manufacturer’s instructions, then diluted with DMEM, 10% FBS, L-glutamine and added to cells after aspirating old media. The next day, the siRNA mix was aspirated and replaced with fresh DMEM, 10% FBS, L-glutamine, collecting samples 72 hr after the start of siRNA treatment. The siRNA were siControl (Luciferase) at 100 nM or a mixture of two MCM3 siRNA (2859 and 2936 at 50 nM each).
siMCM3-2859 5’- augacuauugcaucuucauugdTdT
siMCM3-2936 5’- aacauaugacuucugaguacudTdT

Matson J.P., Dumitru R., Coryell P., Baxley R.M., Chen W., Twaroski K., Webber B.R., Tolar J., Bielinsky A.K., Purvis J.E, & Cook J.G. (2017). Rapid DNA replication origin licensing protects stem cell pluripotency. eLife, 6, e30473.

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siRNA-Mediated Knockdown in NIH/3T3 Cells

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Transfection was performed using DharmaFECT 1 (T-2001-01, Dharmacon) following the manufacturer’s recommendations. Briefly, NIH/3T3 cells were seeded in poly-D-lysine (100 μg mL⁻¹) coated 6-well plates at 15,000 cells cm⁻². After 18 hours, cells were rinsed, incubated with transfection medium, and prepared as follows. A 500 nM solution of siGENOME lamin A/C siRNA (D-001050-01-05, Dharmacon) and a solution of 50x diluted DharmaFECT 1 were made with serum-free DMEM, and then combined in a 1:1 ratio. After incubating for 20 minutes, this solution was added to antibiotic-free complete medium (DMEM + 10% fetal calf serum) in a 1:4 ratio to yield transfection medium. After 72 hours incubation with transfection medium, cells from nine wells were harvested and pooled, then immediately assessed with flow cytometry. A non-transfected control and a non-targeting transfection control using siGENOME non-targeting siRNA #1 (GE: D-001210-01-5) were simultaneously prepared.

Zhang J., Alisafaei F., Nikolić M., Nou X.A., Kim H., Shenoy V.B, & Scarcelli G. (2020). Nuclear mechanics within intact cells is regulated by cytoskeletal network and internal nanostructures. Small (Weinheim an der Bergstrasse, Germany), 16(18), e1907688.

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siRNA-Mediated Functional Screening in C33A2 Cells

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A custom-made siRNA DHARlibrary consisting siGENOME SMARTpools of four siRNAs to each mRNA was ordered from GE Healthcare Dharmacon. Each well in the 96-well plates contained 0.1 nmol of siRNA pool, including one well with a scrambled control siRNA. The siRNA library was transfected in triplicates at three independent occasions into C33A2 cells grown in 96-well plates using DharmaFECT1 (GE Healthcare Dharmacon) according to the instructions of the manufacturer. Cell culture medium was harvested at 72 h posttransfection and sLuc activity was monitored as described above. Additional siRNAs to hnRNP L, hnRNP A1, hnRNP A2B1 and Akt1, as well as scrambled negative control siRNAs, were purchased from GE Healthcare Dharmacon as siRNA SMARTpools to confirm screening results and to perform RNA analysis by transfection of C33A2 cells in 6-well plates. Transfections were conducted with DharmaFECT1 (GE Healthcare Dharmacon) and cell culture medium or cells were harvested 72 h posttransfection.

Kajitani N., Glahder J., Wu C., Yu H., Nilsson K, & Schwartz S. (2017). hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner. Nucleic Acids Research, 45(16), 9654-9678.

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Transfection of miRNA Mimics in Fibroblasts

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Synthetic miRNA mimics (Mimic hsa-miR-1260b and negative control (NC); Gene Design, Ibaraki, Japan) were transfected into Normal Fibroblast (NF) cells at 30 nM using DharmaFECT 1 (Dharmacon, Tokyo, Japan). Twenty-four hours after transfection, NF cells were reseeded. For RNAi experiments, NF cells were transfected with Silencer Select Pre-designed siRNA (Thermo Fisher Scientific) using DharmaFECT 1 (Dharmacon) according to the manufacturer’s protocols.

Morita T., Fujiwara T., Yoshida A., Uotani K., Kiyono M., Yokoo S., Hasei J., Kunisada T, & Ozaki T. (2020). Clinical relevance and functional significance of cell-free microRNA-1260b expression profiles in infiltrative myxofibrosarcoma. Scientific Reports, 10, 9414.

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Lipid-mediated Transfection of TP ODN in HeLa Cells

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To 50 μl of a reduced serum, no antibiotic medium (2% FBS, 1× GlutaMAX, 1× non-essential amino acids and 1× sodium pyruvate) was added a stock solution of a TP ODN in water (Hyclone Cell Molecular biology Grade, Invitrogen) so as to obtain a final concentration of 0.1 μM TP ODN. In an eppendorf tube, 0.25 μl DharmaFECT 1 (purchased from Dharmacon/ThermoFisher, now GE Dharmacon, Lafayette, CO, USA) was added to 50 μl of the reduced serum medium and this solution was added to the TP ODN in the same medium. TP ODN and DharmaFECT 1 (Dharmacon) were mixed and allowed to form a complex at room temperature (20 min). A measure of 275 μl medium was added to the above complex. Medium was removed from 15b HeLa cells in a 96-well plate and 100 μl of the transfection mixture was added to each well. After 24 h of lipid transfection, cells were collected for luciferase assays.

Shang S., Monfregola L, & Caruthers M.H. (2016). Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery. Signal Transduction and Targeted Therapy, 1, 16019-.

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siRNA Knockdown of Ubiquitin Conjugating Enzymes

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RNA interference was performed using siRNA purchased from Dharmacon. HAP1 cells were seeded at 100,000 cells per 6 cm plate and allowed to adhere overnight. Cells were transfected with 25 nM of either nontargeting (Dharmacon D-001810-10), anti-UBE2M (Dharmacon L-004348-00), or anti-UBE2D1 siRNA (Dharmacon L-0093870-00) using 7.5 μL of transfection reagent DharmaFECT 1 (Dharmacon T-2001-02) per well. For quadruple knockdown studies, 12.5 nM of anti-UBE2D1, -UBE2D2 (Dharmacon L-010383-00), -UBE2D3 (Dharmacon L-008478-00), and -UBE2D4 (Dharmacon L-009435-00) siRNA with 15 μL of DharmaFECT1 was used. Transfection reagent was added to OPTIMEM (ThermoFisher 31985070) media and allowed to incubate for 5 min at room temperature. Meanwhile siRNA was added to an equal amount of OPTIMEM. Solutions of transfection reagent and siRNA in OPTIMEM were then combined and allowed to incubate for 30 minutes at room temperature. These combined solutions were diluted with complete DMEM to provide 2 mL per well, and the media exchanged. Cells were incubated with transfection reagents for 48h, at which point the media was replaced with media containing DMSO or 50 μM EN450 and incubated for another 24 h. Cells were then harvested, and protein abundance analyzed by Western blotting.

Siebel C.W., Feng L., Guthrie C, & Fu X.D. (1999). Conservation in budding yeast of a kinase specific for SR splicing factors. Proceedings of the National Academy of Sciences of the United States of America, 96(10), 5440-5445.

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Targeted gene knockdown in PASMCs

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Before transfection, PASMCs were incubated with Opti-MEM-I serum-free medium (Invitrogen) for 3 h before adding 10 nM siRNA lipoplexed with DharmaFECT1 (GE Dharmacon, Lafayette, CO) siRNA/DharmaFECT1 complexes were allowed to form for 20 min at room temperature before being added to the cells. Cells were then incubated with the complexes for 4 h at 37 °C before returning to DMEM/10% FBS overnight. Knockdown efficiency was confirmed by immunoblotting or mRNA expression. The siRNAs used were: ON-TARGETPlus Smartpool oligos for (>x% values represent knockdown at RNA level): ACVR2A (>73%), ADAM10, ADAM17, ALK2 (>84%), BMPR2 (>75%), RELA (>82%, encoding NF-κB p65) or a non-targeting control pool (siCP) (all GE Dharmacon) or oligos targeting FYN (>64%, SASI_Hs01_00195124), HEY1 (>63%, SASI_Hs01_0052320), HEY2 (>63%, SASI_Hs02_00343977), NOTCH2 (>50%, SASI_Hs01_00068801), NOTCH3 (>75%, SASI_Hs01_00101287), SRC (>62%, SASI_Hs01_00112907) or YES (>68%, SASI_Hs01_00086922) from Sigma-Aldrich. For proliferation experiments, we confirmed that the level of knockdown was similar at days 2, 4 and 6 for each target.

Hurst L.A., Dunmore B.J., Long L., Crosby A., Al-Lamki R., Deighton J., Southwood M., Yang X., Nikolic M.Z., Herrera B., Inman G.J., Bradley J.R., Rana A.A., Upton P.D, & Morrell N.W. (2017). TNFα drives pulmonary arterial hypertension by suppressing the BMP type-II receptor and altering NOTCH signalling. Nature Communications, 8, 14079.

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TNBC Cell Line Transfection Assay

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Three human TNBC cell lines were obtained from the Cell Bank of the Chinese Scientific Academy, and their identity was confirmed via human 9-Marker STR DNA profile analysis. Cell were cultured in Minimal Essential Media (MEM) containing 10% fetal bovine serum (FBS, Hyclone) and 2 mM L-glutamine (Invitrogen). Cells were seeded in a six-well plate at a density of 10⁵ cells per well. On reaching 80% confluence, cells were transfected with siRNA or overexpression plasmid using Dharmafect 1 (Dharmacon) by incubating Dharmafect 1 and siRNA or plasmid. siRNA against CERK: UAC AGG CAC AGA GCA GGC A (CERK siRNA#1) and UGC CUG CUC UGU GCC UGU A (CERK siRNA#2) and negative control siRNA (ON-TARGETplus Non-targeting siRNA) were purchased from Dharmacon. CERK was cloned into pCMV3-C-his vector (Sino Biological). Protein expression analysis was performed at 48 h post-transfection. Paclitaxel, cisplatin, LY294002, PP242 and rapamycin were from Selleck, and 10-DEBC was from R&D Systems.

Zhu S., Xu Y., Wang L., Liao S., Wang Y., Shi M., Tu Y., Zhou Y, & Wei W. (2021). Ceramide kinase mediates intrinsic resistance and inferior response to chemotherapy in triple‐negative breast cancer by upregulating Ras/ERK and PI3K/Akt pathways. Cancer Cell International, 21, 42.

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Overexpression and Knockdown of FXN in Cells

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Full-length human FXN cDNA sequence was synthesized (Genewiz) and subcloned into pCDNA3.1 Hygro (Invitrogen). For 293T transfections, cells were seeded at 500,000 cells/well in 6-well plates overnight and then transfected using Fugene 6 (Promega; 3 μl/well) and 1 μg total DNA per well. For fibroblasts, cells were seeded at 200,000 cells/well in 6-well plates and plasmid reverse transfections were carried out immediately using TransIT X2 (Mirus; 5 μl/well) and 2.5 μg total DNA per well. Cells were harvested as noted at the indicated post-transfection time points. siRNA oligo smartpools (Dharmacon) and individual oligos (source and target sequences listed in Supplementary Table 1) were transfected into 293T cells or patient fibroblasts seeded in 6-well plates using Dharmafect I (Dharmacon; 5 μl/well) and the indicated concentration of oligo. Cells were later harvested at the indicated post-transfection time points.

Nabhan J.F., Gooch R.L., Piatnitski Chekler E.L., Pierce B, & Bulawa C.E. (2015). Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin. Scientific Reports, 5, 18251.

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Nanog siRNA Transfection Protocol

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A Nanog siRNA pool (four siRNAs targeting Nanog) and a negative control were purchased from Dharmacon. siRNAs were made to 20 μM in nuclease free water, aliquoted, and stored −80 °C until use. Fibroblasts were seeded into 24 well plates at 9,000 cells per well one day prior to transfection. On the day of transfection siRNAs were diluted to 5 μM in nuclease free water. For each well 5 μl of the working siRNA solution was diluted with 95 μl Optimem-Serum Free media (Gibco, Catalogue number 31985-070). In a separate tube 5 μl of dharmafect-I (Dharmacon, Catalogue number T-2001-02) was diluted with 95 μl Optimem-Serum Free. After a 5 minute incubation the two solutions were combined. After 20 minutes growth media lacking antibiotics was added (800 μl) and the transfection complexes added to the cells.

Wang X., Hodgkinson C.P., Lu K., Payne A.J., Pratt R.E, & Dzau V.J. (2016). Selenium Augments microRNA Directed Reprogramming of Fibroblasts to Cardiomyocytes via Nanog. Scientific Reports, 6, 23017.

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