Packaging plasmids and short hairpin RNA plasmids against NPC1 (sh‐NPC1) or a scrambled control plasmid (sh‐scr; pGFP‐C‐shLenti) were purchased from Origene (Rockville, MD, USA). The plasmids were grown in bacteria and extracted by maxiprep (Macherey‐Nagel, NucleoBond® Xtra Maxi, 740414.10). PCMV and PMD2.G (packaging plasmids) were added with polyethylenimine (PEI) to HEK295T cells, along with sh‐NPC1 or sh‐scr plasmids. Cell medium was replaced with Opti‐MEM, and lentiviral particles were isolated by ultracentrifugation after 48 h of incubation. The pellet was resuspended in PBS and stored at −80°C until use. Lentiviral particles were added to 6 DIV neurons and incubated ON, after which the medium was replaced with fresh medium. Neurons were transfected with the mCherry‐D4 plasmid 33 at 13 DIV with Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. Z‐stack images were acquired using a Nikon A1R+ confocal microscope.
Nucleobond xtra maxi
Manufactured by Macherey-Nagel
Sourced in Germany
NucleoBond Xtra Maxi is a high-performance plasmid DNA purification kit designed for the isolation of high-quality plasmid DNA from bacterial cultures. The kit utilizes a silica-based resin and a modified alkaline lysis procedure to efficiently capture and purify plasmid DNA from cell lysates.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Qiaquick, by Qiagen (1 mentions)
Pureyield, by Promega (1 mentions)
Nucleobond xtra midi, by Macherey-Nagel (1 mentions)
Pei max, by Polysciences (1 mentions)
Stellar competent cells, by Takara Bio (1 mentions)
Pcep4 myc ace2, by Addgene (1 mentions)
Xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Lgk 201, by Toyobo (1 mentions)
Glomax navigator system, by Promega (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Peptone, by Merck Group (1 mentions)
Peptone, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
8 protocols using nucleobond xtra maxi
1
NPC1 Knockdown in Neuronal Cultures
2
Recombinant FHR-1 Protein Production
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FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs). Mutated FHR-1 complementary DNA was cloned in the pCMV mammalian expression vector and purified by NucleoBond Xtra Maxi (740414.10; Macherey Nagel). Mammalian Hek293 freestyle suspension cells (K900001; Fisher Scientific) were transfected following the manufacturer’s instructions and incubated at 37°C with 5% carbon dioxide at 90-rpm agitation. Culture supernatants were collected after 5 days, and FHR-1 proteins were purified and stored as described for the plasma-derived FHR-1 proteins (supplemental Materials and methods, available on the Blood Web site).
3
Cloning and Purification of DNA Plasmids
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DNA was cloned using standard molecular biology procedures (29 (link), 30 (link), 31 (link)) and propagated in a JM109 recA- lacIQ (Zymo Research) strain for purification. Small-scale purifications were done by miniprep (PureYield, Promega) followed by a PCR purification for desalting (QiaQuick, Qiagen). Large-scale purifications were done by midiprep or maxiprep (NucleoBond Xtra Midi or NucleoBond Xtra Maxi, Macherey-Nagel). All plasmids were isolated in stationary phase and sequenced before use.
4
Recombinant Protein Expression in Expi293F Cells
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The recombinant plasmid DNA of all HexaPro-foldon, HexaPro-foldon-I53-50A1 trimer, monomeric hACE2 and 14 mAbs as described above was extracted from bacterial culture of transformed DH5a competent cells in Terrific Broth medium according to manufacturer’s standard protocol (Macherey-Nagel, NucleoBond® Xtra Maxi, Cat#740414.50). Expi293FTM cells were grown in Union 293 medium, diluted to a density of 1.0×106 cells per mL, and then transiently transfected with 1:3 (v/v) containing 1 mg recombinant plasmid:1 mg/ml PEI-MAX (Polyscience, Cat#24765) in Union 293 medium. After 5 days, the cell cultures containing targeted proteins were centrifuged to remove cell debris at 15,000×g for 2 h, and then filtered using a 0.22 µm pore-size vacuum-driven filter.
5
Bacterial Plasmid Transformation and Purification
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Plasmids were transformed into Stellar competent cells (Takara Bio), and transformed cells were plated and grown at 37 °C overnight. Colonies were mini-prepped per the manufacturer’s recommendations (GeneJET, K0502, Thermo Fisher Scientific) and sequence confirmed (Sequetech) and then maxi-prepped per the manufacturer’s recommendations (NucleoBond Xtra Maxi, Macherey-Nagel). Plasmids were sterile filtered using a 0.22-μm syringe filter and stored at 4 °C.
6
Recombinant SARS-CoV-2 Spike Protein Production
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The hACE2 ectodomain (residues 18–615) was fused with the HA signal sequence, a linker containing a 8*HIS-tag, a Twin-Strep-tag and TEV protease cleavage site into vector pCEP4 using Gibson assembly.22 (link) The same strategy was used to create the expression plasmid for wild type SARS-CoV-2 spike receptor binding domain (reference genome Wuhan-HU-1; residues 333–529). Templates used to create the PCR fragments were pTwist-EF1alpha-SARS-CoV-2-S-2xStrep (gift from Nevan Krogan; Addgene plasmid #141382), pCEP4-myc-ACE2 (gift from Erik Procko; Addgene plasmid #141185). Oligonucleotides for Gibson assembly and site-directed mutagenesis were obtained from IDT (Integrated DNA Technologies, Coralville, USA). Single amino acid changes in the RBD (N501Y, E484K, K417N) and combinations thereof were introduced using QuikChange (XL Site-Directed Mutagenesis Kit, Agilent, Santa Clara, USA). Resulting expression plasmids were purified using Nucleobond Xtra maxi, (Macherey-Nagel, Dueren, Germany) and sequence verified.
7
Luciferase Reporter Assay Protocols
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In our reported gene assays, pNL1.2 (#N1001, Promega) was used as a vector. Fragment DNA was inserted into the pNL1.2 vector using Ligation high ver.2 (LGK-201, Toyobo) with KpnI (#R0142S; New England Biolabs, Inc.) and NheI (#R0131S; New England Biolabs) according to the manufacturer's protocol. Transformation was performed using Competent Quick DH5a (DNA-913F; Toyobo), and plasmids were purified using NucleoBond Xtra Maxi (Macherey-Nagel) according to the manufacturer's instructions. Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Cells were assayed after vorinostat or vehicle treatment for 6 hours using the Nano-Glo Luciferase Assay System (N1120; Promega) with the GloMax Navigator System (Promega) according to the manufacturer's instructions. A measurement time of 1 second was used for NanoDLR, and the relative promoter activity was calculated.
8
rHA-ACE Fusion Protein Expression and Purification
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The rHA-ACE fusion proteins were designed by codon optimizing the nucleic acid sequence of the ACE2 ectodomain (amino acids 18-740 of Uniprot ID: Q9BYF1-1) for expression in a human cell line. This was subcloned by GenScript into pcDNA3.1 plasmids containing the WT and KAHQ (NB) variants [32] (link). Nucleic acids encoding a single GGGGS peptide linker connects the ACE2 C-terminal and the rHA N-terminal.
Plasmids were amplified by transformation into TOP10 chemically competent E. coli using heat shock (40 min on ice, 2 min at 42 °C, 5 min on ice) and plated on agar plates (1% peptone (Merck, #82303), 0.5% yeast extract (Fisher Scientific, #BP9727), 0.8% NaCl (Acros Organics, #207790010), 1.5% agar (Sigma, #05040), and 0.1 mg/mL ampicillin (Fisher Scientific, #BP1760)) and cultured overnight. A single colony was selected and grown overnight in a peptone yeast extract broth (3.2% peptone, 2% yeast extract, and 0.5% NaCl) under selection with 0.1 mg/mL ampicillin (Fisher Scientific, #BB176-25). Plasmids were purified using the NucleoBond Xtra Maxi (Macherey-Nagel, #740414) according to the manufacturer's protocol.
Plasmids were amplified by transformation into TOP10 chemically competent E. coli using heat shock (40 min on ice, 2 min at 42 °C, 5 min on ice) and plated on agar plates (1% peptone (Merck, #82303), 0.5% yeast extract (Fisher Scientific, #BP9727), 0.8% NaCl (Acros Organics, #207790010), 1.5% agar (Sigma, #05040), and 0.1 mg/mL ampicillin (Fisher Scientific, #BP1760)) and cultured overnight. A single colony was selected and grown overnight in a peptone yeast extract broth (3.2% peptone, 2% yeast extract, and 0.5% NaCl) under selection with 0.1 mg/mL ampicillin (Fisher Scientific, #BB176-25). Plasmids were purified using the NucleoBond Xtra Maxi (Macherey-Nagel, #740414) according to the manufacturer's protocol.
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