pCMV6-XL5 plasmid (OriGene Technologies, Beijing, China) containing full-length cDNA of human TERT (3.6 kb) [Genbank:NM_198253.2] was amplified in DH5α E. coli strain. The cDNA clone of TERT and GV166 lentiviral vector (GeneChem Co., Ltd., Shanghai, China) were digested by a cocktail of EcoR I and Sal I (New England Biolabs, Ipswich, USA). The subsequent fragments were purified and recombined by T4 ligase (New England Biolabs) and then transformed into DH5α E. coli selecting for ampicillin resistance. The transformants were screened for correct insertion/orientation of the TERT fragment by restriction analysis. GV166 vector not recombined with TERT was used as the control vector. For lentiviral production, the GV166-TERT or control plasmid was co-transfected into 293FT cells with Lenti-Easy Packaging Mix (GeneChem Co., Ltd.) at a 1:3 ratio using Lipofectamine™ reagent (Invitrogen). Forty-eight hours after transfection, the virus-containing supernatant was harvested and stored in aliquots at −80 °C. All cell culture procedures were performed under biosafety level 2 conditions.
Pcmv6 xl5 plasmid
Manufactured by OriGene
Sourced in United States
The PCMV6-XL5 plasmid is a vector designed for the expression of proteins in mammalian cells. It contains a cytomegalovirus (CMV) promoter to drive the expression of the gene of interest, and an XL5 selection marker for identifying and selecting transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (2 mentions)
Pre mir 214 3p, by Thermo Fisher Scientific (2 mentions)
Anti mir 214 3p, by Thermo Fisher Scientific (2 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Lenti easy packaging mix, by Genechem (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Hamilton syringe, by Merck Group (1 mentions)
Total rna purification kit, by Norgen Biotek (1 mentions)
Dharmafect 4, by Thermo Fisher Scientific (1 mentions)
Dharmafect duo transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Ecori, by Thermo Fisher Scientific (1 mentions)
Dpni, by Thermo Fisher Scientific (1 mentions)
Xbai, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nucleo bond xtra plasmid purification kit, by Macherey-Nagel (1 mentions)
Pyrobest dna polymerase, by Takara Bio (1 mentions)
8 protocols using pcmv6 xl5 plasmid
1
Lentiviral Transduction of TERT
2
AAV2-mediated gene delivery in mice
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For AAV2 production, we used a pCMV6-XL5 plasmid (Origene, Rockville, MD, USA) carrying the hNRN1 (NM_016588) cDNA clone. Vector Biolabs (Philadelphia, PA, USA) performed the AAV cis cloning, cis-plasmid preparation, viral packaging, viral purification and GC titration of AAV2-CAG–hNRN1-WPRE. AAV2–GFP with the CAG/CBA promoter was also ordered from Vector Biolabs.
For AAV2 IVT injections, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and 2 μl of AAV2-CAG–GFP or AAV2-CAG–hNRN1 (1010 GC) was injected into the left eye using a Hamilton syringe (Sigma-Aldrich, St. Louis, MO, USA). The contralateral eye was left untreated. To study transduction efficiency, single IVT AAV2 injections were administrated and mice harvested at 2, 3, 4 and 6 weeks post injection (n=4). For studying RGC survival (n=3–7), axonal regeneration (n=4–8) and RGC function (n=6), single AAV2 injections were administered 2 weeks prior to ONC surgery and mice were harvested or tested at 7, 14, 21 and 28 dpc for RGC survival, RGC function and at 28 dpc for axonal regeneration.
For AAV2 IVT injections, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and 2 μl of AAV2-CAG–GFP or AAV2-CAG–hNRN1 (1010 GC) was injected into the left eye using a Hamilton syringe (Sigma-Aldrich, St. Louis, MO, USA). The contralateral eye was left untreated. To study transduction efficiency, single IVT AAV2 injections were administrated and mice harvested at 2, 3, 4 and 6 weeks post injection (n=4). For studying RGC survival (n=3–7), axonal regeneration (n=4–8) and RGC function (n=6), single AAV2 injections were administered 2 weeks prior to ONC surgery and mice were harvested or tested at 7, 14, 21 and 28 dpc for RGC survival, RGC function and at 28 dpc for axonal regeneration.
3
miR-214-3p and CUG-BP1/Survivin Modulation
4
miR-214-3p and CUG-BP1/Survivin Modulation
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Cells were seeded in 6 cm plates at a density of 0.5-1 × 106, a day prior to transfection. For miR transfections, pre-miR-214-3p (12 nM), anti-miR-214-3p (25 nM), or control miR (Ambion, Austin, TX) was diluted in 500μl Opti-MEM I (Invitrogen, Carlsbad, CA) containing 5 μl Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). After 15 min incubation at room temperature (RT), the complex was added to the cells in a final volume of 5 ml of fresh medium. In order to transiently over-express CUG-BP1 and survivin, TE7 cells were transfected with 2 μg of pCMV6-XL5 plasmid containing CUG-BP1 or survivin cDNA (OriGene, Rockville, MD) diluted in 500μl Opti-MEM I containing 5 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
5
Engineered Human Insulin Production
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The bacterial strains employed in this study were obtained from the School of Life Sciences, in the Discipline of Microbiology (University of KwaZulu-Natal, Westville Campus, South Africa). The pCMV6-XL5 plasmid integrated with the gene encoding for human proinsulin was procured from ORI Gene (United States of America (USA). Commercial human insulin protein was obtained from Sigma-Aldrich Inc (Germany) as a control in this study. All restriction enzymes were purchased from Thermo-Scientific, USA.
6
Modulating miR-29a and FOXA2 in Huh7 cells
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Huh7 cells were plated on collagen 1–coated 6-well plates (Becton Dickinson) 1 day before transfection. At ∼70–80% confluency, the cells were transfected with either 10 nmol/L miRIDIAN hsa-miR-29a mimic (Thermo Scientific, Waltham, MA), 10 nmol/L mmu-miR-29a-3p LNA inhibitor (Exiqon, Woburn, MA), or 100 nmol/L ON-TARGETplus human siRNA against FOXA2 (Thermo Scientific) using either DharmaFECT 4 (Thermo Scientific) or Lipofectamine 2000 (Life Technologies, Grand Island, NY) transfection reagent. A human FOXA2 open reading frame (ORF) expression vector containing FOXA2 transcript variant 1 in the pCMV6-XL5 plasmid (OriGene, Rockville, MD) was transfected (1 μg) using DharmaFECT Duo transfection reagent (Thermo Scientific). Forty-eight hours after transfection, total RNA was isolated from the cells using the Total RNA Purification Kit from Norgen.
7
Overexpression of β2 Microglobulin Affects HLA Expression
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Tumour cells were transfected with a human β2 m (NM_004048) expressing, pCMV6-XL5 plasmid (OriGene, Herford, Germany) using Lipofectamine® LTX (HCT116 cells) or Lipofectamine® 3000 (A549 and MCF-7 cells) (Invitogen) and following manufacturer's instructions. Briefly, DNA-lipid complexes were added to cells before incubating for 48 hours at 37oC and 5% CO2 in a humidified atmosphere. Expression of HLA-A,B,C at the cell surface was then determined by flow cytometry. Cells “mock transfected” with a non-encoding plasmid were used as a negative control
8
Generation of CYP2C9 Variant Constructs
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CYP2C9*1 cDNA in pCMV6-XL5 plasmid (Origene, Rockville, MD, USA) was released by digestion with EcoR I and Xba I (Thermo Scientific, Beijing, China), and then subcloned into pcDNA3.1 (±) vector (Invitrogen, Carlsbad, CA, USA). Site-directed mutagenesis to introduce the 430C>T (CYP2C9*2), 449G>A (CYP2C9*8), 980T>C (CYP2C9*31), 1003C>T (CYP2C9*11), and 1075A>C (CYP2C9*3) transitions was performed using pcDNA3.1 (±) carrying human CYP2C9*1 cDNA as the template for polymerase chain reaction amplification by Pyrobest DNA polymerase (TaKaRa, Dalian, China). The specific base transition was introduced into the amplification products by a pair of completely complementary primers containing the substituted base (Supplemental Table 1 ). After incubation with Dpn I (Thermo Scientific) to digest the template and purification, these vectors were transformed into E. coli Top 10 (Tiangen, Beijing, China), and then purified with NucleoBond Xtra plasmid purification Kit (MACHEREY-NAGEL, Germany). Clones carrying the desired mutants were identified by direct DNA sequencing. DNA concentration and quality were evaluated with Nano Drop 2000 UV-Vis Spectrophotometer (Thermo, Wilmington, DE, USA).
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