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E coli stbl2

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The E. coli STBL2 is a high-efficiency competent cell line designed for the cloning and maintenance of difficult-to-clone DNA sequences. It is suitable for the transformation of ligation mixtures and the propagation of plasmids.

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Lab products found in correlation

Miniprep kit, by Qiagen (2 mentions) Quikchange lightning 2 cloning kit, by Agilent Technologies (2 mentions) Dh5 alpha e coli, by Thermo Fisher Scientific (2 mentions) Q5 high fidelity polymerase, by New England Biolabs (2 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Pcrii, by Thermo Fisher Scientific (1 mentions) T4 dna ligase, by Takara Bio (1 mentions)

Close spelling variants of this product

E. coli MAX Efficiency Stbl2 Stbl2™ E. coli host E. coli TOP10 or Stbl2 Stbl2

4 protocols using e coli stbl2

1

Cloning and Sequencing Bunyavirus Genomes

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Complete genome segments were amplified from the cDNA of BTV-1 RSArrrr/01 and BTV-26 KUW2010/02 by PCR using the primers shown in Table 1. The upstream primers contained a T7 promoter sequence and a restriction site for unidirectional cloning, while the downstream primers contained restriction sites for cloning and for linearization at the 3’ end of the segment, as described [53 (link)]. PCR was carried out using a PCR extender system (5 Prime) and the PCR products were double digested with the appropriate restriction enzymes. These were ligated into pGEX-4T-2, digested with the same enzymes, and transformed into competent cells of E. coli STBL2 (Invitrogen). Colonies obtained on ampicillin-containing agarose plates were confirmed by plasmid purification (Qiagen) and DNA sequencing (using the BigDye Terminator v3.1 Cycle Sequencing Kit, Invitrogen). Alternatively, plasmid clones for some segments were synthesized by GeneArt (see results section for details).
Pullinger G.D., Guimerà Busquets M., Nomikou K., Boyce M., Attoui H, & Mertens P.P. (2016). Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells). PLoS ONE, 11(2), e0149709.
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2

Generation of Caspase-3 and Caspase-7 Variants

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Open reading frames (ORF) of human Caspase-3 and Caspase-7 (Source Bioscience) were amplified with primers adding EcoRI and XhoI sequences to 5’ and 3’ ends respectively, digested (Takara), purified (Nucleospin Extract II, Magerey-Nagel), subcloned into pcRII (Invitrogen) by using T4 DNAligase (Takara) and amplified in E. coli Stbl2 (Invitrogen). Then, a SfiI fragment containing the ORF was subcloned in the pEIGW-SK lentiviral vector (kind gift of Dr. Trono, Switzerland). Cysteine to Serine mutants from Caspase-3 and Caspase-7 were obtained using a Site-Directed Mutagenesis System (LifeTechnologies), and their ORF were sequence-verified. Lentiviruses were prepared in the HEK293T packing cell line as described previously [17 (link)]; cardiomyocytes were treated or processed after 4 days of transduction, as described elsewhere [17 (link)].
Cardona M., López J.A., Serafín A., Rongvaux A., Inserte J., García-Dorado D., Flavell R., Llovera M., Cañas X., Vázquez J, & Sanchis D. (2015). Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart. PLoS ONE, 10(6), e0131411.
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3

Molecular Biology Techniques Compendium

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All plasmids were prepared using commercial MiniPrep kits (Qiagen, Hilden, Germany). All enzymes were obtained from New England Biolabs and used per the manufacturer’s recommendations. PCR reactions were carried out using Q5 High-Fidelity Polymerase (New England Biolabs, Ipswich, MA) per the manufacturer’s protocols. Primers were ordered from Genosys (Sigma-Aldirch, Burlington, MA) or Integrated DNA Technologies (Coralville, IA). Except where noted, genomic DNA was extracted using QuikExtract DNA reagent (Lucigen, Middleton, WI). Except where noted, non-viral plasmids were maintained in competent DH5-alpha E. coli, prepared by the Vanderbilt Molecular Cell Biology Resource (MCBR), lentiviral and retroviral plasmids were maintained in Stbl2 E. coli (ThermoFisher, Waltham, MA), and cloned plasmids were initially transformed into competent cells included with kits. Gibson assemblies were carried out using the NEB Gibson Assembly Cloning kit. Site Directed Mutagenesis was carried out using the QuikChange Lightning II cloning kit (Agilent, Santa Clara, CA). PCR cloning was carried out using the NEB PCR cloning kit. All Sanger sequencing was completed by Genewiz, Inc (South Plainfield, NJ).
Reisman B.J., Guo H., Ramsey H.E., Wright M.T., Reinfeld B.I., Ferrell P.B., Sulikowski G.A., Rathmell W.K., Savona M.R., Plate L., Rubinstein J.L, & Bachmann B.O. (2021). Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase. Nature chemical biology, 18(4), 360-367.
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4

Molecular Biology Techniques Compendium

Check if the same lab product or an alternative is used in the 5 most similar protocols
All plasmids were prepared using commercial MiniPrep kits (Qiagen, Hilden, Germany). All enzymes were obtained from New England Biolabs and used per the manufacturer’s recommendations. PCR reactions were carried out using Q5 High-Fidelity Polymerase (New England Biolabs, Ipswich, MA) per the manufacturer’s protocols. Primers were ordered from Genosys (Sigma-Aldirch, Burlington, MA) or Integrated DNA Technologies (Coralville, IA). Except where noted, genomic DNA was extracted using QuikExtract DNA reagent (Lucigen, Middleton, WI). Except where noted, non-viral plasmids were maintained in competent DH5-alpha E. coli, prepared by the Vanderbilt Molecular Cell Biology Resource (MCBR), lentiviral and retroviral plasmids were maintained in Stbl2 E. coli (ThermoFisher, Waltham, MA), and cloned plasmids were initially transformed into competent cells included with kits. Gibson assemblies were carried out using the NEB Gibson Assembly Cloning kit. Site Directed Mutagenesis was carried out using the QuikChange Lightning II cloning kit (Agilent, Santa Clara, CA). PCR cloning was carried out using the NEB PCR cloning kit. All Sanger sequencing was completed by Genewiz, Inc (South Plainfield, NJ).
Reisman B.J., Guo H., Ramsey H.E., Wright M.T., Reinfeld B.I., Ferrell P.B., Sulikowski G.A., Rathmell W.K., Savona M.R., Plate L., Rubinstein J.L, & Bachmann B.O. (2021). Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase. Nature chemical biology, 18(4), 360-367.
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