AAVs carrying hGFAP::Cre and CAG::FLEx-GFP for serotype testing in human islets were as previously described 55 . AAV8-U6-mINS2utr5sg-EGFP-2cut (6.15×10 13 GC/ml) was produced and purified by Penn Vector Core. AAV-DJ-U6-mINS2utr5sg-EGFP-2cut (2.92×10 12 GC/ml), AAV-DJ-U6-hINSin1sg-CopGFP-2cut (1.83×10 13 GC/ml), AAV-DJ-U6-hINSin1sg-CopGFP-1cut (6.02×10 12 GC/ml), and AAV-DJ-nEF-Cas9 (3.83×10 12 GC/ml) were produced and purified as described below. Briefly, recombinant AAVs were produced in 293AAV cells (Cell Biolabs, San Diego, CA). Polyethylenimine (PEI, linear, MW 25,000) was used for transfection of triple plasmids: the pAAV vector constructs, pAAV2/8-RC (Penn Vector Core) or pAAV-DJ (Cell Biolabs) and pHelper (Cell Biolabs). Cells were scrapped in their medium and centrifuged, frozen, and thawed four times by placing it alternately in dry ice-ethanol and 37°C water bath, 72hrs post transfection. AAV crude lysate was purified by centrifugation at 54,000 rpm for 1hr in discontinuous iodixanol gradients with a Beckman SW55Ti rotor. The virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters (Millipore-Sigma, Bedford MA). Virus titers were determined by qPCR according to Addgene protocol.
Millipore amicon ultra centrifugal filters
Manufactured by Merck Group
Sourced in United States
Millipore Amicon Ultra centrifugal filters are laboratory equipment designed for the rapid concentration and purification of biomolecules. They utilize a membrane-based filtration system to separate and concentrate samples during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Phelper, by Cell Biolabs (2 mentions)
Paav dj, by Cell Biolabs (2 mentions)
293aav cells, by Cell Biolabs (2 mentions)
Sw55ti rotor, by Beckman Coulter (2 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Ni nta resin, by Sangon (2 mentions)
Quickrun fast running buffer, by MDBio (2 mentions)
Flexirun premixed gel solution, by MDBio (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Trelief sosoo cloning kit, by Tsingke (1 mentions)
I 5 2 high fidelity master mix, by Tsingke (1 mentions)
Plasmid miniprep kit, by Tsingke (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
6 protocols using millipore amicon ultra centrifugal filters
1
AAV Serotype Evaluation in Human Islets
2
Production and Purification of Recombinant AAVs
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AAVs carrying hGFAP::Cre and CAG::FLEx-GFP for serotype testing in human islets were as previously described.58 (link) AAV8-U6-mINS2utr5sg-EGFP-2cut (6.15 × 1013 GC/mL) was produced and purified by Penn Vector Core. AAV-DJ-U6-mINS2utr5sg-EGFP-2cut (2.92 × 1012 GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-2cut (1.83 × 1013 GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-1cut (6.02 × 1012 GC/mL), and AAV-DJ-nEF-Cas9 (3.83 × 1012 GC/mL) were produced and purified as described below. Briefly, recombinant AAVs were produced in 293 AAV cells (Cell Biolabs, San Diego, CA, USA). Polyethylenimine (PEI, linear, MW 25,000) was used for transfection of three plasmids: the pAAV vector constructs, pAAV2/8-RC (Penn Vector Core) or pAAV-DJ (Cell Biolabs), and pHelper (Cell Biolabs). At 72 h post-transfection, cells were scrapped in their medium, centrifuged, and then frozen and thawed four times by placing alternately in dry ice-ethanol and a 37°C water bath to lyse the cells and release the virus. The resulting AAV crude lysate was purified by centrifugation at 54,000 rpm for 1 h in discontinuous iodixanol gradients with a Beckman SW55Ti rotor. The virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters (Millipore-Sigma, Bedford MA, USA). Virus titers were determined by quantitative PCR (qPCR) according to Addgene protocol.
3
Bacterial Strain Preservation and Cloning
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The strains of E. coli DH5α and BL21(DE3) were preserved by our laboratory. All chemicals and antibiotics were obtained from TCI (Shanghai, China), Solarbio (Beijing, China), Sigma Aldrich (St. Louis, MO, USA) or Thermo Scientific (Shanghai, China). I-5™ 2× High-Fidelity Master Mix and Trelief SoSoo Cloning Kit were obtained from TsingKe (Beijing, China). Plasmid Miniprep Kit from TsingKe was used to prepare plasmid DNA from E. coli DH5α. ClonExpress II One-Step Cloning Kit was purchased from Vazyme (Nanjing, China). The 10× QuickRun™ Fast Running Buffer and FlexiRun™ premixed gel solution for SDS-PAGE were obtained from MDBio (Xinbei, China). Ni–NTA resin used for protein purification was supplied by Sangon Biotech (Shanghai, China). PD-10 desalting columns were purchased from GE Healthcare (Piscataway, NJ, USA). Millipore Amicon Ultra centrifugal filters were obtained from Millipore (Billerica, MA, USA).
4
Bacterial Strain and Plasmid Preservation
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The strains of Escherichia coli DH5α and BL21(DE3) and the plasmid pET28(b) were preserved by our laboratory. All antibiotics and chemicals including TAGs, FFAs, and α-olefins were obtained from Tokyo Chemical Industry (TCI) (Shanghai, China), Solarbio (Beijing, China), Sigma Aldrich (St. Louis, MO, USA), and Thermo Scientific (Shanghai, China). Soybean oil, peanut oil, and olive oil were purchased from local market. Coconut oil and palm oil were obtained from Orifera (Malaysia) and Pythonbio (Guangzhou, China), respectively. The 10 × QuickRun™ Fast Running Buffer and FlexiRun™ premixed gel solution for SDS-PAGE analysis were obtained from MDBio (Xinbei, China). Purification of DNA fragments was performed using a MonPure™ Gel & PCR Clean Kit from Monad (Wuhan, China). Ni–NTA resin used for protein purification was purchased from Sangon Biotech (Shanghai, China). PD-10 desalting columns were supplied by GE Healthcare (Piscataway, NJ, USA). Millipore Amicon Ultra centrifugal filters were bought from Millipore (Billerica, MA, USA).
5
Cell Cultivation and Dengue Virus Propagation
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Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher Scientific) with 10% fetal bovine serum (FBS, Merck, Sigma-Aldrich, USA) was used to cultivate BHK21 [C-13] cells (ATCC CCL-10) and Aedes albopictus clone C6/36 (ATCC CRL-1660) cells. To retain dengue virus (DENV) serotype 2 (PL046), the Aedes albopictus clone cells were exposed to DENV2. DENV was incubated at 28°C with 5% CO2 for five days at a multiplicity of infection (MOI) of 0.01 on C6/36 cell monolayers. Concentrated and filtered viral supernatant was kept in the freezer at −80°C before use, using Millipore Amicon Ultra centrifugal filters (Billerica, MA, USA).
6
Synthesis and Purification of Gold Nanoparticles
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2.1 Materials. All reagents, unless cited otherwise, were purchased from Sigma-Aldrich, and used without any further purification. HAuCl4•3H2O was stored as a 10 mM solution in MilliQ water, at 4 °C and protected from light. The glassware used for gold NPs synthesis were cleaned with aqua regia (HCl (37%) / HNO3 (65%) 3/1). Ultra-pure MilliQ water was used for the preparation of all the aqueous solutions. The concentration of water dispersible NPs was performed by means of Millipore Amicon® Ultra centrifugal filters with a 30 kDa cut-off for CoxFe3-xO4-DMSA NPs and CoxFe3-xO4-DMSA-Au NPs, and with a cut-off of 3 kDa for Au-DMSA NPs. The purification of NPs was performed by using dialysis tubes with a cut-off of 14 kDa. The dropped addition of HAuCl4 to the reaction mixture was performed with the aid of a NE-1010 Higher Pressure Programmable Single Syringe Pump.
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