AAV (serotype 2/8) was produced for gene delivery in vivo53 (link). Briefly, packaging plasmid (pRC-2/8), purified helper plasmid (pHelper), and transfer plasmid (containing the interest cassette) were mixed at a 1:1:1 (w/w/w) ratio and transfected into HEK-293FT cells plated on a 15-cm dish (8 × 106 cells/dish) using PEI. Seventy-two hours after transfection, cells were harvested and resuspended in 3 ml lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl), following by freezing and thawing three times. After addition of MgCl2 (final concentration, 1 mM) and benzonase (final concentration, 50 U ml-1), cell lysis mixtures were incubated at 37 °C for 40 min and centrifuged at 4000 × g for 10 min to collect the supernatant. The supernatant was loaded onto an iodixanol gradient and centrifuged at 350,000 × g for 2 h at 4 °C. The 40% iodixanol fraction was isolated, washed with PBS and concentrated to a final volume 100–200 μL using Amicon Ultra 15 centrifugal filter devices-100 K (Millipore, Bedford, MA). The purified AAV was titered using a quantitative PCR.
Amicon ultra 15 centrifugal filter devices 100 k
Manufactured by Merck Group
Sourced in United Kingdom, Germany, France
The Amicon Ultra 15 centrifugal filter devices-100 K are laboratory equipment designed for the concentration and purification of samples. They feature a 100 kDa molecular weight cut-off membrane that allows the separation of molecules based on size.
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4 protocols using amicon ultra 15 centrifugal filter devices 100 k
1
Production and Purification of Dual-Serotype AAV for In Vivo Gene Delivery
2
Production and Purification of AAV5 Vectors
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Recombinant adenoassociated virus AAV serotype 5 (AAV5) were generated by a triple transfection of HEK293T-derived cell line using polyethylenimine (PEI) transfection reagent and the three following plasmids: pAAV-CMV-mCherry-mU6-shRNA, pXR5 (deposited by Dr Samulski, UNC Vector Core) encoding the AAV serotype 5 capsid and pHelper (Agilent) encoding the adenovirus helper functions. 48 h after transfection, AAV5 vectors were harvested from cell lysate treated with Benzonase (Merck) at 120 U/ml. They were further purified by gradient ultracentrifugation with Iodixanol (OptiprepTM density gradient medium) followed by dialysis and concentration against Dulbecco’s PBS using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Devices 100 K, Millipore). Viral titers were quantified by Real-Time PCR using the LightCycler480 SYBR Green I Master (Roche) and primers targeting mCherry sequence. Titers are expressed as genome copy per milliliter (GC/ml).
3
Extracting and Studying Coral Viromes
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The O. patagonica samples were collected in June 2019 from the marine protected area of Tabarca in Spain (38°09′59´´N, 0°28′56´´W). Three coral samples were transported to the laboratory in a cooler within the next 2 h and immediately processed to obtain the secreted mucus [18 (link)]. Five milliliters of the mucus obtained from the three coral colonies was used to extract the deoxyribonucleic acid (DNA) and to study the natural viral assemblage (hereafter sample MVO5), the supernatant was filtered through 0.2-μm polyethersulfone (PES) filters to eliminate cells and obtain the coral viral fraction. Then, the sample was concentrated to ~200 μl using the Amicon Ultra-15 Centrifugal Filter Devices 100 K (Merck Millipore, Germany. In addition, 5 ml of the coral mucus were incubated at 28°C during 48 h to assess the effect of the increase of seawater temperature on the natural phage community (MVO4, which was also referred to as “stressed” virome) (Fig. 1 ).
4
Recombinant AAV Serotype PHP.eB Production
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Recombinant AAV serotype PHP.eB was generated by a triple transfection of the HEK293T/17 cell line using polyethylenimine (PEI) transfection reagent and the 3 following plasmids: pAAV-CMV-eGFP-H1-shRNA (expressing shRNAs targeting CAG repeats or scrambled, sequences given in Figure 1 B and Figure 4 A), pUCmini-iCAP-PHP.eB [18 (link)] (encoding the AAV serotype PHP.eB capsid), and pHelper (Agilent, Santa Clara, CA, USA) (encoding the adenovirus helper functions). After 48 h of transfection, rAAV vectors were harvested from the cell lysate and treated with Benzonase (Merck Millipore 101697, Molsheim, France) at 100 U/mL. They were further purified by gradient ultra-centrifugation with Iodixanol (OptiprepTM density gradient medium, Sigma-Aldrich, Saint-Quentin-Fallavier, France)) followed by dialysis and concentration against Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich) using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Devices 100K, Merck Millipore). Viral titers were quantified by real-time PCR using the LightCycler480 SYBR Green I Master (Roche Diagnostics, Meylan, France) and primers targeting the eGFP sequence. Titers were expressed as viral genome copies per milliliter (vgc/mL).
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