Plasmid maxiprep kit

Manufactured by Qiagen

Sourced in Germany

The Plasmid Maxiprep Kit is a laboratory product designed for the purification of large quantities of high-quality plasmid DNA from bacterial cultures. The kit utilizes a modified alkaline lysis method and a silica-based membrane technology to efficiently isolate plasmid DNA.

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26 protocols using plasmid maxiprep kit

Cloning and Expressing Human ISG15 in HepG2.2.15 Cells

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As previously described [15 (link)], human full-length ISG15 gene was generated using pOTB7-ISG15 plasmid DNA (MGC clones; Open Biosystems) as template, and the resulting PCR product was cloned into a pcDNA4/HisMax TOPO TA expression vector (Invitrogen, USA). The sequences of the primers are 5′-ATGGGCTGGGACCTGACGGTG-3′ (forward) and 5′-TTAGCTCCGCCCGCCAGGCTC-3′ (reverse), respectively. Plasmid Maxiprep Kit (Qiagen, German) was used to prepare plasmid DNA for transfection.
HepG2.2.15 cells with integrated full-length HBV genome were kindly provided by Professor Bo Qin (Chongqing Medical University, China), as previously described [17 (link)], and were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA), supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 100 IU mL⁻¹ penicillin (Gibco, USA), 100 IU mL⁻¹ streptomycin (Gibco, USA), and 1 mg mL⁻¹ G418 (Invitrogen, USA) in 5% CO₂ at 37°C.

Li Y., Li S., Duan X., Chen Y., Jiao B., Ye H., Yao M, & Chen L. (2016). Interferon-Stimulated Gene 15 Conjugation Stimulates Hepatitis B Virus Production Independent of Type I Interferon Signaling Pathway In Vitro. Mediators of Inflammation, 2016, 7417648.

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Generating Saturated Libraries of NPHV-Rand and HCV-Rand

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To generate saturated libraries of NPHV-Rand and HCV-Rand containing all possible combinations of nucleotides in the randomized region, we constructed bacterial transformation libraries with at least 10 times more colonies than the combination of all possible nucleotides (4^6 = 4096). Briefly, we introduced randomized seed sites into NPHV-SL1/miRBR and HCV by PCR with RU-O-21135 and RU-O-17131 or RU-O-21134 and RU-O-17131, respectively. After restriction digest with NotI and KpnI, the PCR product was ligated into the parental plasmid and transformed into DH10B with electroporation transformation. After transformation, cells were revived in 500 μl SOC medium. 2 μl were spread on P10 LB medium to count colonies. To minimize potential bias of bacterial growth, bacteria were spread onto 2xP500 LB plates. After overnight incubation, cells were scraped into 20 ml LB medium and plasmids were purified with Plasmid Maxi-prep kit (Qiagen) without further expansion.

Yu Y., Scheel T.K., Luna J.M., Chung H., Nishiuchi E., Scull M.A., Echeverría N., Ricardo-Lax I., Kapoor A., Lipkin I.W., Divers T.J., Antczak D.F., Tennant B.C, & Rice C.M. (2017). miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence. PLoS Pathogens, 13(10), e1006694.

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Genome-wide shRNA screen for fluvastatin targets

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A549 cell line, stably transduced with the RNAi Consortium (TRC1) shRNA library [27 (link)] was treated with fluvastatin (US Biologicals) over 12 days. Genomic DNA (gDNA) was amplified and shRNA populations hybridized onto custom Affymetrix Gene Modulation Array Platform (GMAP) arrays. Differences between shRNA abundances over 12 days were assessed using the Bayes Factor [29 (link)]. For generation of stably expressing shRNA cell lines, DNA from bacterial glycerol stocks of shRNAs from the TRC1 library was amplified using E.coli and purified (Qiagen Plasmid Maxiprep kit). Lentiviral particles were generated by calcium phosphate transfection of sub-confluent (50–60%) HEK293TV cells with 10 μg of TRC pLKO.1 puro shRNA construct, 5 μg each of pMDG1.vsvg, pRSV-Rev and pMDLg/pRRE constructs. Lentiviral particles were collected 24 and 48 hours later, filtered though a 0.45 μm filter and stored at −80°C. Parental cell lines were infected with lentiviral particles containing the indicated shRNAs and puromycin selected (48 hrs, 2 μg/ml of puromycin).

Pandyra A.A., Mullen P.J., Goard C.A., Ericson E., Sharma P., Kalkat M., Yu R., Pong J.T., Brown K.R., Hart T., Gebbia M., Lang K.S., Giaever G., Nislow C., Moffat J, & Penn L.Z. (2015). Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death. Oncotarget, 6(29), 26909-26921.

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Generation of sub-genomic CRISPR library

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Our sub-genomic CRISPR library was cloned following previous methods (Shalem et al., 2014 (link)) using previously characterized sgRNAs (Wang et al., 2014 (link)). Five unique sgRNA inserts along with 50 non-targeting controls were synthesized by Custom Array of the form:
The oligo pool was diluted 1:10 in water and amplified using NEB Phusion Hotstart Flex enzyme master mix and the following primers:
Inserts were cleaned with Axygen PCR clean-up beads (1.8x; Fisher Scientific) and resuspended in molecular biology grade water. lentiCRISPRv2 (Addgene ID# 52961) was digested with BsmBI (Thermo Fisher) for 2 hours at 37°C. The large ~ 13 kB band was gel extracted after size-selection on a 1% agarose gel. Using 100ng of cut lentiCRISPRv2 and 40ng of sgRNA library inserts, a 20μL Gibson assembly reaction was performed (30min, 50°C). After Gibson assembly, 1μL of the reaction was transformed into electrocompetent Lucigen cells and spread on LB-ampicillin plates and incubated overnight. After counting dilution plates to ensure library coverage, colonies were scraped and combined for plasmid extraction using a Plasmid Maxiprep kit (Qiagen).

Anderson G.R., Winter P.S., Lin K.H., Nussbaum D.P., Cakir M., Stein E.M., Soderquist R.S., Crawford L., Leeds J.C., Newcomb R., Stepp P., Yip C., Wardell S.E., Tingley J.P., Ali M., Xu M., Ryan M., McCall S.J., McRee A.J., Counter C.M., Der C.J, & Wood K.C. (2017). A landscape of therapeutic cooperativity in KRAS mutant cancers reveals principles for controlling tumor evolution. Cell reports, 20(4), 999-1015.

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DNA Isolation from Whole-Blood Samples

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The Plasmid Maxiprep Kit (QIAGEN) was used to isolate DNA according to the manufacturer’s instructions from frozen whole-blood samples of patients and control subjects.

Zhao L.P., Alshiekh S., Zhao M., Carlsson A., Elding Larsson H., Forsander G., Ivarsson S.A., Ludvigsson J., Kockum I., Marcus C., Persson M., Samuelsson U., Örtqvist E., Pyo C.W., Nelson W.C., Geraghty D.E, & Lernmark Å. (2016). Next-Generation Sequencing Reveals That HLA-DRB3, -DRB4, and -DRB5 May Be Associated With Islet Autoantibodies and Risk for Childhood Type 1 Diabetes. Diabetes, 65(3), 710-718.

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Generation of E6AP HECT Domain Library

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The gene of the E6AP HECT domain was PCR amplified from pET-E6AP with primers WY11 and WY12. The amplified fragment was double-digested with NheI and XhoI, and cloned into pCTCON2 plasmid to generate pCTCON2-E6AP HECT. To generate E6AP library in the pCTCON2 plasmid, the E6AP HECT domain gene was PCR amplified with WY13 and WY14 to incorporate randomized codons at residues 651, 652, 653, 654, and 656. The PCR fragment amplified with WY13 and WY14 was combined with fragment amplified with WY11 and WY12 to assemble the HECT library gene by overlapping extension with primers WY11 and WY12. The amplified library gene was digested with NheI and XhoI, and cloned into the pCTCON2 vector. Transformation of the plasmid library into XL1 blue electro-competent cells afforded a library of 2.0 × 10⁸ in diversity, large enough to cover all the mutants with randomized residues replacing D651, D652, M653, M654 and T656 in the E6AP HECT domain. Transformed XL1 blue cells were plated on LB-ampicillin plates (LB agar supplemented with 100 μg mL⁻¹ ampicillin) and allowed to grow at 37 °C overnight. Colonies growing on the plate were scraped and the library DNA was extracted with the Plasmid Maxiprep Kit (Qiagen).

Wang Y., Liu X., Zhou L., Duong D., Bhuripanyo K., Zhao B., Zhou H., Liu R., Bi Y., Kiyokawa H, & Yin J. (2017). Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer. Nature Communications, 8, 2232.

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Lentiviral Plasmid Construction: SV40 Large T Antigen

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The temperature-sensitive large T antigen SV40 sequence, SVU19tsa58, was digested from pZipSVU19tsa58 plasmid (a gift from Parmjit Jat, UCL, UK) using the BamH1 restriction site as previously described^{10 (link),11 (link)}. The sequence was ligated into the pLVX Puro lentiviral plasmid (Clontech, CA) using the same restriction sites and sequenced to confirm correct orientation. Plasmid DNA was amplified in One Shot Stbl3 E. coli (Thermo-Fisher Scientific) and purified using a Plasmid Maxiprep kit (Qiagen).

Prideaux M., Wright C.S., Noonan M.L., Yi X., Clinkenbeard E.L., Mevel E., Wheeler J.A., Byers S., Wijenayaka A.R., Gronthos S., Sankar U., White K.E., Atkins G.J, & Thompson W.R. (2021). Generation of two multipotent mesenchymal progenitor cell lines capable of osteogenic, mature osteocyte, adipogenic, and chondrogenic differentiation. Scientific Reports, 11, 22593.

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Barcoded Luciferase Reporter Library Construction

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After generating the TRE unit/barcode dictionaries, fragments containing a firefly luciferase CDS and one of three minimal promoters – thymidine kinase (minTK), the minimal promoter of Promega’s pGL4 plasmid suite (minProm), or cytomegalovirus (minCMV) – were created in a modified pDonor_eGP2AP_RC via Gibson Assembly, digested with KpnI and XbaI, and then ligated into the barcoded plasmid library replicates using the KpnI and XbaI restriction enzyme sites. Each replicate received one of the three minimal promoters.
Plasmid libraries for transfection were prepared by inoculating 100 mL of 2X YT media containing 50 ug/mL kanamycin with 200 uL of bacterial glycerol stocks and incubating at 30 degrees C with shaking at 225 rpm overnight. Cultures were pelleted and plasmids were purified using a Plasmid Maxiprep Kit (Qiagen) according to manufacturer’s protocol. Plasmid preparations were then combined at equimolar ratios. TRE-MPRA libraries are available from Addgene under deposit number 82594.

Zahm A.M., Owens W.S., Himes S.R., Rondem K.E., Fallon B.S., Gormick A.N., Bloom J.S., Kosuri S., Chan H, & English J.G. (2023). Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters. bioRxiv.

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Construction of Rsp5 HECT Domain Mutant Library

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The HECT domain gene of Rsp5 was PCR-amplified from pET-Rsp5 by PCR, double-digested with NheI and XhoI, and ligated with pCTCON2 plasmid digested with the same restriction enzymes. Overlapping extension PCR was used to construct a library of Rsp5 HECT domain genes with randomized mutations replacing residues Val606, Leu607, Asp608, Thr610 and Asn632. PCR primers WY7 and WY8 were designed to have NNK codons substituting the codons for the residues to be randomized in the library. These primers paired with WY 5 and WY 6 respectively to amplify fragments of HECT domain gene with randomized codons at residue positions 606, 607, 608, 610 and 632. The two PCR fragments were then assembled by an overlapping extension PCR with primers WY5 and WY6. The amplified PCR fragment was digested with NheI and XhoI, and ligated with the pCTCON2 vector. Transformation of the plasmid library into XL1 Blue competent cells afforded a library of 2.0 × 10⁸ in size, large enough to cover all the mutants with five randomized residues. Transformed XL1 Blue cells were plated on LB-ampicillin plates (LB agar supplemented with 100 μg/mL ampicillin) and allowed to grow at 37°C overnight. Colonies appeared on the plate were scraped and the library DNA was extracted from the scraped cells with the Plasmid Maxiprep Kit (Qiagen).

Wang Y., Fang S., Chen G., Ganti R., Chernova T.A., Zhou L., Duong D., Kiyokawa H., Li M., Zhao B., Shcherbik N., Chernoff Y.O, & Yin J. (2021). Regulation of the Endocytosis and Prion Chaperoning Machineries by Yeast E3 Ubiquitin Ligase Rsp5 as Revealed by Orthogonal Ubiquitin Transfer. Cell chemical biology, 28(9), 1283-1297.e8.

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Murine D6/2 Rescue Plasmids: Generation and Characterization

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The murine D6/2 rescue plasmids: pT7-D6/2-VP2, pT7-D6/2-VP3, pT7-D6/2-VP4, pT7-D6/2-VP6, pT7-D6/2-VP7, pT7-D6/2-NSP1, pT7-D6/2-NSP2, pT7-D6/2-NSP3, and pT7-D6/2-NSP5 were prepared as described previously (29 (link)) while pT7-SA11-VP1 and pT7-SA11-NSP4 were originally made by Dr. Takeshi Kobayashi (Research Institute for Microbial Diseases, Osaka University, Japan) (26 (link)) and obtained from Addgene. The C3P3-G1 plasmid (44 (link)) was kindly provided by Dr. Philippe H Jaïs. To generate pT7-D6/2-NSP3-Nluc (accession number: ON738554), which encodes a full-length Nluc gene (GenBank: KM359774.1) and the self-cleaving P2A peptide gene of porcine teschovires-1, the P2A-Nluc gene cassette was amplified by PCR and inserted between nucleotides in the NSP3 gene via Gibson assembly (NEBuilder HiFi DNA Assembly kit). Purification of all the plasmids was performed using QIAGEN Plasmid Maxiprep kit per the manufacturer’s instructions.

Zhu Y., Sánchez-Tacuba L., Hou G., Kawagishi T., Feng N., Greenberg H.B, & Ding S. (2022). A recombinant murine-like rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission. Frontiers in Immunology, 13, 911024.

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