Plasmid prep kit

Manufactured by Qiagen

Sourced in United States

The Plasmid Prep Kit is a laboratory tool designed to purify plasmid DNA from bacterial cultures. It provides a streamlined process for isolating and concentrating plasmid DNA for various downstream applications such as cloning, sequencing, and molecular biology experiments.

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Plasmid kits (15 citations) Prep Plasmid Midi Kit (9 citations) Mini-prep plasmid extraction kit (8 citations) Endo-Free plasmid Maxi prep Kit (6 citations) Maxi-prep plasmid extraction kit (6 citations) Mini-prep plasmid isolation kit (5 citations) Plasmid purification maxi prep kit (3 citations) Midi Prep Plasmid DNA Extraction kit (3 citations) Plasmid Mega Prep Kit (3 citations) Endo Free Plasmid Giga-prep Kit (3 citations)

6 protocols using plasmid prep kit

Toxicity Assays of Inorganic Compounds

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Sodium arsenite (NaAsO₂) and sodium dichromate dehydrate (Na₂Cr₂O₇) were from Sigma (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), gentamicin, and L-glutamine were from Invitrogen (Carlsbad, CA). RNeasy mini kit and plasmid prep kit were from Qiagen (Valencia, CA, USA). M-MLV reverse transcriptase was from Promega (Madison, WI). Oligo (dT)_20, AccuPrime Taq DNA polymerase high fidelity and pGEM-T easy cloning vector were from Invitrogen (Carlsbad, CA). Luciferase assay system was from Promega (Fitchburg, WI). Antibodies against p53 and phospho-p53 were from Cell Signaling Tech (Danvers, MA). Antibody against p21 antibody was from Santa Cruz (Santa Cruz, CA).

Park Y.H., Kim D., Dai J, & Zhang Z. (2015). Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis. Toxicology and applied pharmacology, 287(3), 240-245.

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Reagents and Materials for Cell Studies

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Sodium dichromate dehydrate (Na₂Cr₂O₇) was from Sigma (St Louis, MO). Cisplatin was from Enzo Life Sciences (Farmingdale, NY). Dulbecco’s modified Eagle’s medium (DMEM), Defined keratinocyte serum-free medium, fetal bovine serum (FBS), and Oligo (dT)₂₀ and AccuPrime Taq DNA Polymerase High Fidelity were from Invitrogen (Carlsbad, CA). Bradford Protein Assay Reagent was from Bio-Rad (Hercules, CA). RNeasy Mini kit and plasmid prep kit were from Qiagen (Valencia, CA). M-MLV reverse transcriptase was from Promega (Madison, WI). Perfecta Sybr Green Fastmix was from Quanta Biosciences (Gaithersburgh, MD). 5-(and -6)-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) and CellROX deep red reagent were from Molecular Probes (Eugene, OR). Antibodies against NQO1, GAPDH, and β-actin were obtained from Santa Cruz (Santa Cruz, CA). Antibody against FBP1 was from Sigma (St Louis, MO). Antibody against SOD2 was from Millipore (Billerica, MA). Antibodies against Notch1, p21, cyclin D1, Bcl-2, Bcl-xL, PARP, AP1/c-jun, cleaved caspase 3, and cleaved caspase 9 were from Cell Signaling Tech (Danvers, MA). Enhanced chemiluminescence reagent was from Amersham (Pittsburgh, PA). Matrigel was from BD Biosciences (San Jose, CA).

Dai J., Ji Y., Wang W., Kim D., Fai L.Y., Wang L., Luo J, & Zhang Z. (2017). Loss of fructose-1,6-bisphosphatase induces glycolysis and promotes apoptosis resistance of cancer stem-like cells: an important role in hexavalent chromium-induced carcinogenesis. Toxicology and applied pharmacology, 331, 164-173.

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Protein Signaling Pathway Assay

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Apigenin, dimethyl sulfoxide (DMSO), polybrene, and MG132 were from Sigma (St Louis, MO). RNeasy mini kit and plasmid prep kit were from Qiagen (Valencia, CA). CellTiter 96® AQueous non-radioactive cell proliferation assay kit and M-MLV reverse transcriptase were from Promega (Madison, WI). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), puromycin, and Oligo (dT)₂₀ AccuPrime Taq DNA polymerase high fidelity were from Invitrogen (Carlsbad, CA). Leibovitz's L-15 medium was from American Type Culture Collection (ATCC) (Manassas, VA). Enhanced chemiluminescence (ECL) kit was from Thermo Scientific (Rockford, IL). Matrigel basement membrane matrix was from BD Biosciences (Sparks, MD). Antibodies against p-FAK^Tyr397, FAK, p-Src^Tyr527, Src, p-CrkL^Tyr207, CrkL, p-Akt^Ser473, and Akt were from Cell Signaling Technologies (Beverly, MA). Antibodies against NEDD9 and β-actin were from Santa Cruz Biotechnology (San Diego, CA). Antibody against CK20 was from Abcam (Cambridge, MA).

Dai J., Van Wie P.G., Fai L.Y., Kim D., Wang L., Poyil P., Luo J, & Zhang Z. (2016). Downregulation of NEDD9 by apigenin suppresses migration, invasion, and metastasis of colorectal cancer cells. Toxicology and applied pharmacology, 311, 106-112.

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Competent Escherichia coli Transformation

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Competent Mach1-T1 Escherichia coli. were transformed with plasmid DNA and inoculated in LB broth overnight for amplification. Plasmids were extracted using a Qiagen Plasmid Prep kit following the manufacturer’s protocol.

Gada K.D., Chang M., Chandrashekar A., Plant L.D., Noujaim S.F, & Logothetis D.E. (2022). Mechanism of PKCε regulation of cardiac GIRK channel gating. Proceedings of the National Academy of Sciences of the United States of America, 120(1), e2212325120.

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Constructing V5-tagged human SM22 and mutants

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pCMV6-XL5-hSM22 was purchased from Origene Inc (#sc118118). To construct the C-terminal V5 tagged human SM22 and its truncation mutants, an EcoRI-XhoI-BamHI-KpnI-V5-XbaI fragment was subcloned into EcoRI and XbaI sites of pcDNA3.1. The resulting construct was cleaved with HindIII and XhoI followed by insertion of the PCR fragments containing the full length V5-tagged human SM22 or its truncation mutants. Clones were verified by sequencing (Genewiz Technologies, INC).
The pCGN-SRF plasmid and the luciferase reporter driven by the 4xCArG boxes from the fos promoter were generous gift from Dr. Eric Olson [28 (link), 29 (link)]. The 1.4kb mouse Egr3 promoter (upstream of the ATG) containing the CArG box (CCATATATGG) was PCR cloned into the XhoI/HindIII sites of pGL3-basic luciferase vector (Promega). About 2kb rat Nik promoter (upstream of the ATG) containing the CArG box (CCAACAATGG) was PCR cloned into the NheI/HIII sites of pGL3-basic luciferase vector (Promega). The CArG box mutant (TTAACAATT) was subsequently generated as the Nik2kbCArGmut-luc. All constructed luciferase reporters containing the promoter fragment or the CArG box mutant were verified by sequencing (Genewiz Technologies, INC). All plasmids DNA were prepared for transfection using the plasmid Prep kit (Qiagen).

Dai X., Thiagarajan D., Fang J., Shen J., Annam N.P., Yang Z., Jiang H., Ju D., Xie Y., Zhang K., Tseng Y.Y., Yang Z., Rishi A.K., Li H.J., Yang M, & Li L. (2017). SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells. PLoS ONE, 12(12), e0190191.

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Multicopy Suppressor Screen in Yeast

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The multicopy suppression screen was performed using a YEP13 library (2µ, LEU2 vector) containing 5-20kb inserts of yeast genomic DNA (Nasmyth and Reed, 1980) . The YEP13 pooled library was transformed into vma21 and vma13 vma21 double mutants and plated onto media lacking leucine to select for plasmid transformation. Approximately 10,000 colonies were then replica plated onto YPG media. For colonies that grew, the plasmid was isolated from yeast using a Qiagen plasmid prep kit (with glass beads added to lysis buffer) and the plasmid was transformed into E. coli, isolated (Qiagen) and sequenced with primers flanking the insertion site to identify the genomic region contained on the plasmid. For validation, FET4 was cloned with ~400bp of the upstream promoter into the pAG425 (resulting plasmid named pBMW182a) backbone using the Yeast Gateway System Vectors (obtained from Addgene) (Alberti et al., 2007) .

Chen K.L., Ven T.N., Crane M.M., Brunner M.L.C., Pun A.K., Helget K.L., Brower K., Chen D.E., Doan H., Dillard-Telm J.D., Huynh E., Feng Y., Yan Z., Golubeva A., Hsu R.A., Knight R., Levin J., Mobasher V., Muir M., Omokehinde V., Screws C., Tunali E., Tran R.K., Valdez L., Yang E., Kennedy S.R., Herr A.J., Kaeberlein M., & Wasko B.M. (2020). Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging inSaccharomyces cerevisiae.

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