Pgem 3z vector

Manufactured by Promega

Sourced in United States

The pGEM-3Z vector is a general-purpose cloning and expression vector. It contains a multiple cloning site, promoters, and other features that allow for the insertion and expression of DNA fragments. The core function of the pGEM-3Z vector is to facilitate the cloning and manipulation of DNA sequences.

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PGEM-3Z plasmid vector PGEM-3Z PGEM vector

17 protocols using pgem 3z vector

Cloning and Sequencing of DNA Fragments

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Cloning was done as per Sambrook & Russell [35 ]. Selected DNA pools were cloned at the EcoRI- HindII site in the pGEM-3Z vector (Promega). Sequencing was performed in the Life Sciences Core Facility at the University at Albany.

Mallik P.K., Shi H, & Pande J. (2017). RNA aptamers targeted for human αA-crystallin do not bind αB-crystallin, and spare the α–crystallin domain. Biochemical and biophysical research communications, 491(2), 423-428.

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Synthesis and Characterization of DMD ZnF Mutants

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The H19 sequence was synthesized by GenScript and cloned into the pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies). An expression vector encoding the wild-type sequence of the human DMD ZnF domain was purchased from the shRNA and ORF core of MD Anderson Cancer Center, and the coding regions were subcloned into the Gateway™ pET-DEST40 vector for mammalian expression and Gateway™ pET-DEST42 vector for prokaryotic expression (Invitrogen). All single-point and deletion mutations were generated using QuikChange™ Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). DMD ZnF Y/F-A mutant substitutes tyrosine (Y) or phenylalanine (F) to alanine (A) at sites Y3326, F3332, Y3334, and F3341. Recombinant DMD WT and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific). Plasmid transfections were performed using Lipofectamine3000 (Life Technologies) or electroporation using the 4D-Nucleofector™ System (Lonza) according to the manufacturer’s instructions.

Li Y., Zhang Y., Hu Q., Egranov S.D., Xing Z., Zhang Z., Liang K., Ye Y., Pan Y., Chatterjee S.S., Mistretta B., Nguyen T.K., Hawke D.H., Gunaratne P.H., Hung M.C., Han L., Yang L, & Lin C. (2021). Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects. Genome Medicine, 13, 137.

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Characterization of LINC00301 RNA Interactions

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LINC00301 was constructed into the pGEM-3Z vector (P2151, Promega) using KpnI (R3142, New England Biolabs) and NheI (R3131, New England Biolabs) restriction enzyme cutting site. Then, pGEM-3Z-LINC00301 was cut into liner DNA using KpnI and NheI restriction enzyme separately. Sense and antisense of LINC00301 RNA were transcribed using MEGAscript™ T7 Transcription Kit (AM1334, Invitrogen, USA) and MEGAscript™ SP6 Transcription Kit (AM1330, Invitrogen, USA) in vitro, respectively. The transcribed LINC00301 was purified by RNA Clean & Concentrator-5 (R1013, Zymo Research, USA). Purified RNAs were biotin-labeled with the Pierce™ Biotin 3′ End DNA Labeling Kit (89818, Thermo Scientific, USA). Positive/negative control and biotinylated RNAs were mixed and incubated with A549 or SPC-A-1 cell lysates at NT2 buffer. Subsequently, the preclear magnetic beads were added to each binding reaction and incubated with rotation at RT. Ultimately, the magnetic beads were washed twice by NT2 buffer and digested by proteinase K (P8107, Promega), and then protein/RNA was isolated for western blot and qRT-PCR assay.

Sun C.C., Zhu W., Li S.J., Hu W., Zhang J., Zhuo Y., Zhang H., Wang J., Zhang Y., Huang S.X., He Q.Q, & Li D.J. (2020). FOXC1-mediated LINC00301 facilitates tumor progression and triggers an immune-suppressing microenvironment in non-small cell lung cancer by regulating the HIF1α pathway. Genome Medicine, 12, 77.

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SARS-CoV-2 Quantification Standard Development

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Table S1A lists the primers and probes used in this study, including primer and probe sets for the detection of SARS-CoV-2 (CDC-N1, CDC-N2, E_Sarbeco), PMMoV, and MS2 bacteriophage. The primers and probe for amplifying the luciferase (luc) gene for the internal amplification control (IAC) are also listed.
The SARS-CoV-2 quantification standard was created by inserting the envelope gene (NCBI: 43740570) and the nucleocapsid gene (NCBI: 43740575) of SARS-CoV-2 into the pGEM-3z vector (Promega Corp.) using the BamH I site. The plasmid was subsequently linearized using Xba I. The linearized plasmid was in-vitro transcribed using T7 RiboMAX™ Large Scale RNA Production System (Promega Corp.) to create Positive Control RNA. The linearized plasmid DNA containing the N and E genes, or the in-vitro transcribed RNA were quantified using droplet digital PCR and used as a quantification control. Linear quantification DNA and positive control RNA for PMMoV and MS2 were generated and quantified using the same methods described above.

Mondal S., Feirer N., Brockman M., Preston M.A., Teter S.J., Ma D., Goueli S.A., Moorji S., Saul B, & Cali J.J. (2021). A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2. The Science of the Total Environment, 795, 148834.

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Overexpression Plasmid Construction and Cell Transfection

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To construct the lncMREF, p300 and Smarca5 overexpression plasmids, the full‐length or coding sequences of lncMREF, p300 and Smarca5 were cloned into the pcDNA3.1 plasmid (Addgene, USA). For in vitro transcription assays, full‐length and truncated fragments of lncMREF were cloned into the pGEM‐3Z vector (Promega, USA). Full‐length sequences of lncMREF, p300 and Smarca5 were amplified using specific F/R primers (Supplementary Materials, Table S3). For cell transfection, we transfected C2C12 myoblasts, human skeletal muscle myoblasts, and mouse myogenic progenitor cells with approximately 4 μg plasmid using 9 μl Lipofectamine 2000 (Invitrogen, USA) in each well of a 6‐well plate.

Lv W., Jiang W., Luo H., Tong Q., Niu X., Liu X., Miao Y., Wang J., Guo Y., Li J., Zhan X., Hou Y., Peng Y., Wang J., Zhao S., Xu Z, & Zuo B. (2022). Long noncoding RNA lncMREF promotes myogenic differentiation and muscle regeneration by interacting with the Smarca5/p300 complex. Nucleic Acids Research, 50(18), 10733-10755.

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Quantification of Fas, FasL, and DcR3 Expression

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The cDNA fragments for human Fas (360 bp; nt +137 to +496), human FasL (377 bp; nt +256 to +632), human DcR3 (389 bp; nt +386 to +774) and human glyceraldehyde-3-phosphate dehydrogenase (115 bp) were derived from human oesophageal cancer cells by RT-PCR cloning and inserted into the pGEM 3Z vector (Promega Corp., Madison, WI, USA). The recombinant plasmids were linearized with the appropriate restriction enzymes and used as templates for in vitro transcription of [³²P]- or [³⁵S]-labeled antisense cRNA probes, which were used in the RNase protection assay (RPA) or in situ hybridization (ISH) experiments, respectively.

Kanbayashi C., Koyama Y., Ichikawa H., Sakata E., Hasegawa M., Toshikawa C., Manba N., Ikarashi M., Kobayashi T., Minagawa M., Kosugi S.I, & Wakai T. (2014). Amplification of Genomic DNA for Decoy Receptor 3 Predicts Post-Resection Disease Recurrence in Breast Cancer Patients. World Journal of Oncology, 5(1), 14-23.

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Molecular Cloning and Expression of DMD

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H19 DNA sequences were synthesized by GenScript and cloned into pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies) or pMS2 vector for mammalian expression. The full-length human or mouse DMD and human DMD zinc finger domain sequence was obtained from MDACC shRNA and ORFeome Core and Addgene. Gateway™ pET-DEST42 vector (Invitrogen) was used for prokaryotic expression of human DMD c-termini. Mammalian full-length DMD, zinc finger domain of DMD, or H19 wild type and mutant vectors were constructed by subcloning the corresponding gene sequences into the SFB-tagged expression vector (provided by J. Chen, MD Anderson Cancer Center, USA) using the Gateway system (Life Technologies). All single-point and deletion mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Scramble siRNA and siRNA targeting TRIM63 were obtained from Santa Cruz Biotechnology. Plasmid transfections were performed using Lipofectamine3000 (Life Technologies), or electroporation using the 4D-Nucleofector^™ System (Lonza) according to manufacturer’s instructions. Recombinant DMD wild type and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific).

Zhang Y., Li Y., Hu Q., Xi Y., Xing Z., Zhang Z., Huang L., Wu J., Liang K., Nguyen T.K., Egranov S.D., Sun C., Zhao Z., Hawke D.H., Li J., Sun D., Kim J.J., Zhang P., Cheng J., Farida A., Hung M.C., Han L., Darabi R., Lin C, & Yang L. (2020). LncRNA H19 Alleviates Muscular Dystrophy Through Stabilizing Dystrophin. Nature cell biology, 22(11), 1332-1345.

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Analytical Sensitivity of MERS-CoV RT-iiPCR Assays

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The analytical sensitivity of the singleplex MERS-CoV RT-iiPCR assays was determined by using in vitro transcribed (IVT) RNA. Briefly, the sequences of the ORF1a and upE regions of the MERS-CoV (nucleotides 27361 to 27596 and nucleotides 11137 to 11339, respectively; https://www.ncbi.nlm.nih.gov/nuccore; accession number NC_019843) were synthesized, cloned into the pGEM-3Z vector (Promega, Madison, WI) downstream of the T7 promoter sequence, and subsequently used as the template in in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit (Ambion/Life Technologies, Grand Island, NY). Residual DNA was removed using the Ambion Turbo DNA-free kit (Applied Biosystems, Grand Island, NY). Concentration of RNA was measured by a NanoDrop1000 Spectrophotometer (NanoDrop Technologies, Houston, TX). Single-use IVT RNA aliquots were stored at −80°C. The analytical sensitivity of the RT-iiPCR was determined using a dilution series (10⁰ to 10⁸ molecules/reaction) of the IVT RNA. The concentration of the IVT RNA/μL was calculated as described previously.³² (link)

Go Y.Y., Kim Y.S., Cheon S., Nam S., Ku K.B., Kim M., Cho N.H., Park H., Alison Lee P.Y., Lin Y.C., Tsai Y.L., Thomas Wang H.T, & Balasuriya U.B. (2017). Evaluation and Clinical Validation of Two Field–Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome–Coronavirus. The Journal of Molecular Diagnostics : JMD, 19(6), 817-827.

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Biotinylated lncRNA Synthesis and Purification

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The HPR lncRNA sequence was cloned into the pGEM-3Z vector (Promega) for in vitro transcription using Biotin RNA Labelling Mix (Roche) and MEGAscript^® Transcription Kit (Life Technologies). Biotinylated RNAs were purified by RNA Clean & Concentrator™-5 (Zymo Research).

Zhang S., Liang S., Wu D., Guo H., Ma K, & Liu L. (2021). LncRNA coordinates Hippo and mTORC1 pathway activation in cancer. Cell Death & Disease, 12(9), 822.

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IVT RNA Synthesis for qPCR Analytical Sensitivity

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Target-specific in vitro transcribed (IVT) RNA was synthesized in order to determine the analytical sensitivity of each multiplex RT-qPCR assay as previously described [45 (link),46 ,47 (link)]. Inserts containing the target regions of each assay (flanked by PstI and HindIII restriction enzymes) were chemically synthesized and cloned into the multiple cloning site of the pGEM^®-3Z vector (Promega, Madison, WI, USA).

Thieulent C.J., Carossino M., Peak L., Wolfson W, & Balasuriya U.B. (2023). Multiplex One-Step RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Enteric Viruses of Dogs and Cats. Viruses, 15(9), 1890.

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