The following DNA constructs were used in this study: Lifeact–mRuby (pN1-Lifeact-mRuby), PA-GFP–actin (pC1 PA-GFP-γ-actin, pC1 PA-GFP β-actin) and EGFP–actin (pEGFP-C1 EGFP γ-actin, pEGFP-C1 EGFP β-actin). DNA constructs were prepared using Endotoxin-free Maxi Prep kits (Qiagen).
Endotoxin free maxi prep kits
Manufactured by Qiagen
Sourced in China
The Endotoxin-free Maxi Prep kits are a set of laboratory equipment designed for the large-scale purification of plasmid DNA. The kits utilize a silica-based membrane technology to efficiently capture and purify plasmid DNA from bacterial cultures, providing a high-quality and endotoxin-free product.
Automatically generated - may contain errors
Lab products found in correlation
Dh5a competent cells, by Thermo Fisher Scientific (2 mentions)
Anti s, by Sino Biological (1 mentions)
Pvivo2 plasmid, by InvivoGen (1 mentions)
Not 1 restriction digest, by New England Biolabs (1 mentions)
Aavpro kit, by Takara Bio (1 mentions)
293ft cell line, by Thermo Fisher Scientific (1 mentions)
Stbl3 escherichia coli, by Thermo Fisher Scientific (1 mentions)
Qiaamp minelute virus spin kit, by Qiagen (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
7 protocols using endotoxin free maxi prep kits
1
Fluorescent Actin Constructs for Live-Cell Imaging
2
SARS-CoV-2 S/N Protein Expression Plasmid
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The SARS-CoV-2 S/N protein-encoding gene, containing an N-terminal Kozak sequence (GCCACC) followed by an initiation codon (ATG), was synthesized using a mammalian-optimized codon (GenScript Co., Nanjing, China). It was then cloned into the expression vector pcDNA3.1 (+) via EcoRI and XbaI digestion and named pS/pN (DNA vaccines) (Figure 1 A). The pE/pM protein was constructed and identified as described previously [15 (link)]. Vaccines were prepared using endotoxin-free Maxiprep kits (Qiagen, Beijing, China), and sequences were confirmed using Sanger DNA sequencing. The expression of the S/N protein was confirmed using western blotting and anti-S (Sino Biological, Beijing, China)/anti-N antibodies diluted at 1:1000. These experiments were conducted as described previously [15 (link),18 (link)].
3
Plasmid Selection for In Vivo Gene Expression
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An enhanced green fluorescent protein (GFP) expressing plasmid was from Aldevron (pgWiZ; Fargo, ND, USA). This plasmid was used for some in vitro transfection experiments and although produces sustained GFP expression it can induce immune responses in vivo (Chamarthy et al., 2003 (link); Grønevik et al., 2005 (link); Rose et al., 2014 (link)). Hence for in vivo work a bicistronic pVIVO2 plasmid (9.6 kb) was purchased from Invivogen (San Diego CA, USA). pVIVO2 includes a SV40 DNA targeting signal (DTS) for improved nuclear entry with cytosine and guanine separated by only one phosphate (CpG) motifs removed from the plasmid backbone to reduce immune reactions in vivo (Davies et al., 2012 (link)). pVIVO2 also contains two human ferritin composite promoters, FerH (heavy chain) and FerL (light chain) combined with SV40 and CMV enhancers for GFP and LacZ expression, respectively. Competent Escherichia coli cells were transformed with pgWiZ or pVIVO2 plasmids and purified using Endotoxin Free Maxi Prep Kits (Qiagen) as per the manufacturer’s instructions.
4
Actin Visualization Constructs
5
Cre and Fluorescent Protein Plasmids
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pCAG-Cre, pCALNL-EGFP, pCALNL-DsRed are a generous gift from CL Cepko and are currently available through Addgene (plasmids 13775, 13770, 13769). All plasmids were grown in DH5a competent cells (Life Technologies) and purified using endotoxin-free maxiprep kits (Qiagen)45 (link).
6
Generating rAAV6-FOXP3 Viral Particles
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All FOXP3 homology donors were cloned into pAAV-MCS AAV vectors (Agilent Technologies) containing AAV inverted terminal repeats. Cloning was performed using Not I restriction digest [New England Biolabs (NEB)] followed by ligation with T4 DNA ligase (NEB). Plasmid preparation was performed by transforming plasmids into Stbl3 Escherichia coli (Life Technologies) and extracting plasmid DNA with Endotoxin-Free Maxi Prep kits (Qiagen). For rAAV production, pAAV-FOXP3 plasmids were cotransfected with rAAV6 helper plasmid DNA into the 293FT Cell Line (Life Technologies). After 72 hours, rAAV6-FOXP3 viral particles were extracted using the AAVpro kit (Clontech, Takara) according to the manufacturer’s instructions. The viral stocks were titered using quantitative PCR (qPCR) with primers and probe annealing to the inverted terminal repeats (ITRs). Briefly, the rAAV genomic DNA was isolated using QIAamp MinElute Virus Spin Kit (Qiagen), qPCR was performed on the Roche LightCycler 480, and viral titer (vector genomes per microliter) was calculated using a standard curve generated from a circular pAAV-MCS-donor plasmid of known concentration.
7
Cre-LoxP Fluorescent Reporter Plasmids
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pCAG-Cre, pCALNL-EGFP, pCALNL-DsRed are a generous gift from T. Matsuda & C. L. Cepko and are currently available through Addgene (plasmids 13775, 13770, 13769). pEGFP-N1 was from Clontech. mCherry was a generous gift from Dr. Roger Tsien. All plasmids were grown in DH5a competent cells (Life Technologies) and purified using endotoxin-free maxiprep kits (Qiagen).
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