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Stbl3 e coli

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Stbl3 E. coli is a laboratory strain of Escherichia coli bacteria. It is commonly used for the propagation and maintenance of plasmid DNA in molecular biology and genetic engineering applications.

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Lab products found in correlation

Quickchange lightning kit, by Agilent Technologies (2 mentions) Bigdye terminator v3, by Thermo Fisher Scientific (2 mentions) Seqstudio genetic analyzer, by Thermo Fisher Scientific (2 mentions) Endofree plasmid maxi kit, by Qiagen (2 mentions) Plko 1, by Addgene (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Zero blunt pcr cloning kit, by Thermo Fisher Scientific (1 mentions) Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Lenti icas9 neo, by Addgene (1 mentions) Lentiguide, by Addgene (1 mentions) 5 alpha competent e coli, by New England Biolabs (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Custom oligos, by Integrated DNA Technologies (1 mentions) Pcmv vsv g, by Addgene (1 mentions) T4 polynucleotide kinase, by New England Biolabs (1 mentions) Pmdlg prre, by Addgene (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Prsv rev, by Addgene (1 mentions)

25 protocols using stbl3 e coli

1

SCA17 Allele Expansion Analysis

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Spinocerebellar ataxia type 17 allele CAG/CAA-repeat tracts from 30 patients from this cohort were PCR amplified using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers which flank the CAG/CAA-repeat region and introduce restriction enzyme cleavage sites. The primers used were TBPx3 BamHI Forward (5′-ATGTTTggatccTCCACAGGGTGCCATGAC-3′) and TBPx3 XhoI Reverse (5′-GGTTTGctcgagACACGAAGTACTCACTGC-3′), with restriction sites indicated in lower case. The PCR reactions were processed and cloned into a pcDNA3.1(+) vector similar to previously described for SCA1 alleles (Menon et al., 2013 (link)) or pCR-Blunt using the Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific). Clone plasmids were propagated in Stbl3 E. coli (Thermo Fisher Scientific) (genotype F−mcrB mrr hsdS20(rB−, mB−) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(StrR) xyl-5 λ−leumtl-1). Plasmids were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).
Nethisinghe S., Lim W.N., Ging H., Zeitlberger A., Abeti R., Pemble S., Sweeney M.G., Labrum R., Cervera C., Houlden H., Rosser E., Limousin P., Kennedy A., Lunn M.P., Bhatia K.P., Wood N.W., Hardy J., Polke J.M., Veneziano L., Brusco A., Davis M.B, & Giunti P. (2018). Complexity of the Genetics and Clinical Presentation of Spinocerebellar Ataxia 17. Frontiers in Cellular Neuroscience, 12, 429.
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2

Recombinant ALKBH5 Plasmid Generation

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The wild type ALKBH5-CDS and mutant ALKBH5-CDS were PCR-amplified from pF RT/TO/HIS/FLAG/HA-ALKBH5 plasmid (38073, addgene) and pFLAG CMV5.1-ABH 5-H204A (kindly provided by Dr. Chuan He), and then cloned into the pCDH lentivir al vector (CD513B-1, SBI, Mountain View, CA) using XbaI and BamHI enzyme sites. The TRC shRNAs targeting human ALKBH5 (shA5-#1: TRCN0000291838; shA5-#2: TRCN0000291769), mouse Alkbh5 (shA5-#a: TRCN0000201776; shA5-#b: TRCN00001 92524) were purchased from Sigma-Aldrich, the non-targeting control (pLKO.1) was fr om addgene. The inducible shRNA plasmids (TRIPZ-shA5-#3: V2THS_173653; TRIPZ-shA5-#4: V2THS_173654), as well as the non-targeting control shRNA, were all purchased from GE Dharmacon. The Lenti-iCas9-neo (doxycycline-inducible Cas9-EGFP ve ctor) and lenti-guide (gRNA expression vector) were purchased from Addgene. Lenti-s gALKBH5 was constructed as previously described (Ran et al., 2013 (link)). Stbl3™ E.coli (C7373–03, Thermo Fisher Scientific) and 5-alpha Competent E. coli (C29871, New E ngland Biolabs) were used in transformation.
Shen C., Sheng Y., Zhu A., Robinson S., Jiang X., Dong L., Chen H., Su R., Yin Z., Li W., Deng X., Chen Y., Hu Y.C., Weng H., Huang H., Prince E., Cogle C.R., Sun M., Zhang B., Chen C.W., Marcucci G., He C., Qian Z, & Chen J. (2020). M6A demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia. Cell stem cell, 27(1), 64-80.e9.
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3

Isolation of pUC19 Plasmid DNA

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DNA solutions were prepared from Stbl3 E. coli (ThermoFisher, Waltham, MA) transformed with the pUC19 plasmid. Bacteria were grown in an incubator at 37C for two days in LB Broth containing 100μM ampicillin prior to harvesting and centrifugation. The resulting bacteria were then lysed and DNA was isolated using a DNEasy Blood and Tissue Kit (Qiagen). DNA samples were analysed using a NanoDrop (Thermo Scientific) to determine concentration and verify purity.
Haber J.M., Gascoyne P.R, & Sokolov K. (2017). Rapid Real-Time Recirculating PCR Using Localized Surface Plasmon Resonance (LSPR) and Piezo-electric Pumping. Lab on a chip, 17(16), 2821-2830.
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4

CRISPR-Cas9 sgRNA Design and Lentiviral Production

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Three sgRNA sequences targeting Napa (5′-CTTGAACATGTTCGCCGCTC-3′, 5′-AGATTGCTCATAGTGGGCGA-3′, 5′-TGCTGGGAACGCTTTCTGCC-3′) and Ppp2cb (5′-TCTCGCACAGCGTCCGCACT-3′, 5′-ATACGAACTACCTATTCATG-3′, 5′- CGCAATATTGTGATGCGCTC-3′) were designed using CHOPCHOP (version 3) [21 (link)]. Custom oligos (Integrated DNA Technologies, Coralville, IA, USA) were phosphorylated in vitro with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA), annealed by heating/cooling in a thermocycler, ligated into the BsmBI site of pSECC (Addgene # 60820) with T4 DNA Ligase (New England Biolabs, Ipswich, MA, USA), and plasmid was purified from Stbl3 E. coli (Thermofisher, Waltham, MA, USA). Lentiviral particles for each individual sgRNA construct and a scrambled control (5′-GCGAGGTATTCGGCTCCGCG-3′) [22 (link)] were generated by Lipofectamine Plus/LTX-mediated transfection of 293FT cells with pSECC, pCMV-VSV-G (Addgene #8454), pMDLg/pRRE (Addgene #12251), and pRSV-Rev (Addgene #12253). The supernatant containing viral particles was collected at 48 h post-transfection.
Doyle T.B., Muntean B.S., Ejendal K.F., Hayes M.P., Soto-Velasquez M., Martemyanov K.A., Dessauer C.W., Hu C.D, & Watts V.J. (2019). Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D1 and D2 Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening. Cells, 8(11), 1468.
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5

HIV Virus Production and Immune Cell Assays

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Both cell lines (HEK293T/17 cells) and primary human immune cells were used to make stocks of infectious HIV virus and create target/effectors cells for co-culture assays. For all primary human samples, peripheral blood mononuclear cells (PBMCs) were collected by Ficoll gradient separations from buffy coat samples, cryopreserved and stored at −150°C for future use. All HIV and CAR T cell plasmids were grown up in Stbl3 E.coli (ThermoFisher, Cat#C737303).
Clayton K.L., Mylvaganam G., Villasmil-Ocando A., Stuart H., Maus M.V., Rashidian M., Ploegh H.L, & Walker B.D. (2021). HIV-infected macrophages resist efficient NK cell-mediated killing while preserving inflammatory cytokine responses. Cell host & microbe, 29(3), 435-447.e9.
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6

Generation and Validation of IL-10RA Plasmids

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pcDNA3.1( +) vector coding for wild-type or mutant IL-10RA were purchased from GenScript and expanded in Stbl3 E. coli (Thermo Fisher) according to standard protocols. Plasmids were purified using the E.Z.N.A.® Endo-Free Plasmid Maini Kit (Omega bio-tek). Following expansion in bacterial cultures and plasmid purification, all IL-10RA sequences were re-validated by Sanger sequencing (Source BioScience) to exclude mutagenesis during propagation.
Aschenbrenner D., Ye Z., Zhou Y., Hu W., Brooks I., Williams I., Capitani M., Gartner L., Kotlarz D., Snapper S.B., Klein C., Muise A.M., Marsden B.D., Huang Y, & Uhlig H.H. (2022). Pathogenic Interleukin-10 Receptor Alpha Variants in Humans — Balancing Natural Selection and Clinical Implications. Journal of Clinical Immunology, 43(2), 495-511.
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7

CRISPRi Guide RNA Cloning and Lentivirus Production

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Guide RNAs for CRISPRi experiments were chosen from (Horlbeck et al. 2016 (link)). Guides were cloned into the LRG2.1 mCherry vector (Addgene; Cat. No. 108099) using a BsmBI digestion as previously described (Giuliano et al. 2019 ). Plasmids were transformed in Stbl3 E. coli (Thermo Fisher; Cat. No. C737303) and sequenced to confirm the presence of the correct gRNA sequence. A dCas9-KRAB construct (Addgene; Cat. No. 85969) was used to knock down target gene expression. Guide RNA sequences are listed in Table S1B.
Lentivirus was generated using calcium phosphate transfection as previously described (Chang et al. 2013 (link)). Supernatant was harvested at 2 to 3 intervals 48 to 72 hr post-transfection by filtering through a 0.45 μm syringe, and then frozen at −80° C for later use or supplied directly to cells with 4 μg/mL polybrene.
Vasudevan A., Baruah P.S., Smith J.C., Wang Z., Sayles N.M., Andrews P., Kendall J., Leu J., Chunduri N.K., Levy D., Wigler M., Storchová Z, & Sheltzer J.M. (2020). Single chromosomal gains can function as metastasis suppressors and promoters in colon cancer. Developmental cell, 52(4), 413-428.e6.
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8

HIV Variant Plasmid Construction

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Plasmids encoding HIV variants were constructed by inducing the mutations on pNL4.3 [26 (link)] (the plasmid was obtained from M. Martin through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health) using the QuickChange Lightning kit (Agilent Technologies, Santa Clara, CA, USA) with mutagenesis primers (Table S2) as previously described [16 (link)]. Mutagenesis was confirmed by Sanger DNA sequencing using the BigDye terminator v.3 (Thermo Fisher) with SeqStudio Genetic Analyzer (Thermo Fisher), and plasmids were maintained in Stbl3 E. coli (Thermo Fisher). The plasmid purification was carried out using the EndoFree Plasmid Maxi Kit (Qiagen).
Yang J., Hao M., Khan M.A., Rehman M.T., Highbarger H.C., Chen Q., Goswami S., Sherman B.T., Rehm C.A., Dewar R.L., Chang W, & Imamichi T. (2021). A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing. Viruses, 13(11), 2331.
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9

CRISPR Plasmid Generation for Experiments

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Guide RNAs for CRISPR experiments were designed with Benchling (www.benchling.com). Guides were cloned into the Lenti-Cas9-gRNA-GFP vector (Addgene #124770) or LRCherry2.1 (Addgene #108099) using a BsmBI digestion as previously described96 (link). Plasmids were transformed in Stbl3 E. coli (Thermo Fisher Scientific, cat. no. C737303) and sequence-verified to confirm the presence of the correct gRNA. CRISPR gRNA sequences are listed in Table S5A.
Girish V., Lakhani A.A., Scaduto C.M., Thompson S.L., Brown L.M., Hagenson R.A., Sausville E.L., Mendelson B.E., Lukow D.A., Yuan M.L., Kandikuppa P.K., Stevens E.C., Lee S.N., Salovska B., Li W., Smith J.C., Taylor A.M., Martienssen R.A., Liu Y., Sun R, & Sheltzer J.M. (2023). Oncogene-like addiction to aneuploidy in human cancers. bioRxiv.
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10

Construction of HIV Variant Plasmids

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Plasmids encoding HIV variants were constructed by site-directed mutagenesis on pNL4.3 [33 (link)] (the plasmid was obtained from M. Martin through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Site-directed mutagenesis was performed using a QuickChange Lightning kit (Agilent Technologies, Santa Clara, CA, USA) with mutagenesis primers (Table S1), as previously described [22 (link)]. RH- or IN-deleted constructs were created by inverting PCR using Pfu II Taq polymerase (Agilent Technology) with deletion primers (Table S1). Mutagenesis was confirmed by Sanger DNA sequencing using the BigDye terminator v.3 (Thermo Fisher) with SeqStudio Genetic Analyzer (Thermo Fisher), and the plasmids were maintained in Stbl3 E. coli (Thermo Fisher). The plasmid purification was carried out using the EndoFree Plasmid Maxi Kit (Qiagen, Germantown, MD, USA).
Imamichi T., Chen Q., Hao M., Chang W, & Yang J. (2022). The C-Terminal Domain of RNase H and the C-Terminus Amino Acid Residue Regulate Virus Release and Autoprocessing of a Defective HIV-1 Possessing M50I and V151I Changes in Integrase. Viruses, 14(12), 2687.
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