miR-641 mimic/mimic NC and specific siRNAs against AP1G1, the pcDNA3.1 vector targeting AP1G1 and the empty vector (Genechem, China) were transfected into the EC cell lines via Lipofectamine 3000 (Thermo Fisher, USA).
Empty vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
An empty vector is a DNA molecule that does not contain any inserted genetic material. It serves as a basic tool in molecular biology and genetic engineering, providing a platform for the cloning and manipulation of genetic sequences. The empty vector provides the necessary components, such as origins of replication and antibiotic resistance markers, to facilitate the introduction, propagation, and selection of recombinant DNA constructs in host cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Fugene 6, by Promega (1 mentions)
Peg ittm virus precipitation solution, by System Biosciences (1 mentions)
Purelink quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Trans lentiviral packaging system, by Thermo Fisher Scientific (1 mentions)
Pgl3 basic plasmid, by Promega (1 mentions)
Amaxa cell line nucleofector kit, by Lonza (1 mentions)
8 protocols using empty vector
1
Modulating AP1G1 Expression in ECs
2
Establishing Stable Pim-Overexpressing Prostate Cancer Cell Lines
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The human androgen-independent prostate epithelial adenocarcinoma cell line PC-3 (American Type Culture Collection) was maintained in Roswell Park Memorial Institute (RPMI) -1640 medium, supplemented with 10% fetal bovine serum, L-glutamine and antibiotics. To prepare PC-3-derived cell lines stably overexpressing human Pim family members, PC-3 cells were transfected with pcDNA3.1/V5-His-C-based expression vectors for Pim-1 or Pim-3 (kindly provided by Markku Varjosalo, University of Helsinki, Finland) or mock-transfected with the empty vector (Thermo Fisher Scientific, Waltham, MA, USA). In addition, all cells were cotransfected with the pCMV-Td-Tomato plasmid (Clontech Laboratories Inc., Mountain View, CA, USA) to be used as a fluorescent follow-up marker. All transfections were performed with Fugene 6 (Promega, Madison, WI) in 3:1 ratio to DNA. After an overnight incubation, positively transfected cells were enriched by antibiotic selection with G418 (Fisher Scientific, Geel, Belgium), first using 300 μg/ml for 48 h and thereafter 500 μg/ml for 14 days. Medium was changed every day during the selection. After selection, maintenance of the transfected plasmids in the stable cell lines generated from pools of cells was ensured by supplementing culture medium with 200 μg/ml of G418.
3
Lentiviral Knockdown of Dck, Nfkb1, Trp53
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The Open Biosystems' shRNA TRC constructs for Dck, 25382 (KD1) and 25383 (KD2), Nfkb1 (9514 and 9511), Trp53 (12359 and 12360), GFP and empty vector (Thermo Fisher Scientific Inc.), were provided in E. coli and plated on carbenicillin media to isolate single clones. Next, the plasmids containing the shRNA constructs were isolated from the E. coli using the InvitrogenTM PureLink® Quick Plasmid Miniprep Kit (Life Technologies Corporation). The plasmids were then transfected into Open Biosystems' packaging cells, TLA-HEK293T, using the Open Biosystems' Trans-Lentiviral™ Packaging System (Thermo Fisher Scientific Inc.). The TLA-HEK239T cells were maintained in the recommended growth media. Viral particles were then collected and concentrated using PEG-itTM Virus Precipitation Solution (System Biosciences, Mountain View, CA). The viral particles were transduced into the B117P and B140P cell lines by adding virus (MOI of 100) and 8 μg/ml of polybrene to the cells and incubating for 2 hours at 37°C followed by spinoculation (30 min, 300 g).
4
Generating Plasmids and Lentiviral Constructs
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The pmCherry C1 human WIP plasmid was purchased from Addgene (#29573). To generate the pcDNA3.1-β-catenin plasmid, we amplified the full-length β-catenin gene by RT-PCR from H1299 cell cDNA and cloned it into pcDNA3.1 vector (V790, invitrogen, USA). To generate the WIP gene promoter-driven luciferase reporter plasmid, the WIP gene upstream sequences (−1691 to +28, −266 to +28) were cloned into PGL3-basic plasmid (Promega, Madison, WI, USA) respectively. The pLX304-Blast V5-PD-L1 plasmid and Empty Vector (#OHS6895, Thermo, USA) were transfected into HEK-293T cells with lenti-HIV expression packaging kit (LT001, GeneCopoeia, Guangzhou, China) to produce lentivirus. At 48 h after transfection, lentivirus was used to transduct H1299 and A549 cells. After 72 h, 1 µg/ml or 8 µg/ml Blastcidin S was used for selecting drug-resistant cells.
5
ROBO4 Overexpression and miR-138 Modulation
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The pcDNA3.1-based ROBO4 overexpression vector (pc-ROBO4) was generated via inserting the sequence of ROBO4 in the vector, and the empty vector (Thermo Fisher Scientific, Waltham, MA, USA) was utilized as negative control (pc-NC). MiR-138 mimic (5′-AGCUGGUGUUGUGAAUCAGGCCG-3′), and mimic negative control (miRNA NC; 5′-CGAUCGCAUCAGCAUCGAUUGC-3′) were provided via iGeneBio (Guangzhou, China). HRMECs were transfected with 800 ng vectors or 30 nM mimics using Lipofectamine 3000 (Thermo Fisher Scientific) for 24 hours.
6
Modulating Colorectal Cancer Chemoresistance
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KLF12 overexpression vector (pcDNA-KLF12) was based on pcDNA3.1 vector. The empty vector (Thermo Fisher) was exploited as negative control (pcDNA-NC). siRNA for circ_0071589 (si-circ_0071589, 5ʹ-UGACUAUGACAGUGUCAGCUU-3ʹ), siRNA negative control (si-NC, 5ʹ-AACAGUCGCGUUUGCGACUGG-3ʹ), miR-526b-3p mimic (5ʹ-GAAAGUGCUUCCUUUUAGAGGC-3ʹ), mimic negative control (miR-NC, 5ʹ-CGAUCGCAUCAGCAUCGAUUGC-3ʹ), miR-526b-3p inhibitor (anti-miR-526b-3p, 5ʹ-GCCUCUAAAAGGAAGCACUUUC-3ʹ), and inhibitor negative control (anti-miR-NC, 5ʹ-UGAGCUGCAUAGAGUAGUGAUUA-3ʹ) were synthesized via RiBoBio (Guangzhou, China). These constructed vectors or oligonucleotides were transfected into HCT116/CDDP and LOVO/CDDP cells using Lipofectamine 3000 reagent (Thermo Fisher) for 24 h.
7
Stable Cell Line Generation
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MDA-MB-231 and SKBR3 cell lines (obtained from ATCC) were cultured as reported previously [10 (link)]. Generation of stable cell lines was performed as described previously by electroporation using the Amaxa® cell line nucleofector® kits (LONZA, Walkersville, MD, USA). Plasmids employed for the production of stable cell lines were: pcDNA3.1/Hygro-RANK-c and empty vector (Invitrogen, Carlsbad, CA, USA) [10 (link)].
8
Transfection of E2F1 and Mutant Plasmids
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cDNA 3.1 Plasmid encoding E2F1 and its mutant (E138) were kindly provided by Dr. Doug Cress. Cells were seeded in 6-well plates and transfected with 2 μg plasmids. After 6 hours, the medium with plasmid was removed and cells were incubated with fresh medium for two days. An empty vector (invitrogen) was used as control.
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