DH10B Competent E. coli cells (ThermoFisher Scientific, #EC0113) were used for routine cloning and plasmid propagation. They were cultured in standard LB medium or plated on agar plates supplemented with appropriate antibiotic.
E coli dh10b competent cells
Manufactured by Thermo Fisher Scientific
E. coli DH10B competent cells are a strain of Escherichia coli bacteria that have been genetically modified to increase their competence for transformation with exogenous DNA. They are commonly used in molecular biology laboratories for the cloning and propagation of plasmid DNA.
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5 protocols using e coli dh10b competent cells
1
Routine Cloning with DH10B E. coli
2
Routine Cloning with DH10B E. coli
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DH10B Competent E. coli cells (ThermoFisher Scientific, #EC0113) were used for routine cloning and plasmid propagation. They were cultured in standard LB medium or plated on agar plates supplemented with appropriate antibiotic.
3
Generation of Recombinant Avian Herpesvirus
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A DEVC-KCE infectious clone was generated with a method modified from the generation of BAC from the DEV 2085 strain [9 (link)]. Briefly, co-transfection of DNA from DEVC-KCE and pDEVgc-pHA2 was conducted on primary CEFs (24 h) to allow for an insertion of mini-F sequences in lieu of gC. After green plaques were observed under UV light (488 nm), a homogeneous population of mini-F recombinant DEVC-KCE (DEVC-KCE-miniF) was obtained by three rounds of picking and plating on CEFs and transferred into E. coli DH10B competent cells (Invitrogen) by electroporation. Positive clones with chloramphenicol resistance were examined through RFLP with EcoR I and BamH I to select a clone of DEVC-KCE, which was then electroporated into E. coli GS1783 [9 (link)] for further genetic manipulation of the DEV genome. The resulting clone (pDEVC-KCE) was confirmed through RFLP. Next, gC was restored by homologous recombination as described previously [9 (link)]. A homogenous population of gC-recovered virus (DEVC-KCEgCR) was purified by picking and plating of the nonfluorescent plaques under UV light (488 nm) and verified by PCR and sequencing using a pair of primers (DEV gC flanking F and DEV gC flanking R; Table 1 ).
4
Generation of Infectious HVT-miniF Clones
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The HVT Fc-126 infectious clone was generated with the method modified from that used for the generation of BAC [32 (link)]. Briefly, cotransfection of DNA of HVT Fc-126 and pUC19-H1-H2-miniF was conducted on primary CEFs (24 h) to allow for insertion of mini-F sequences in lieu of glycoprotein C (gC). After green plaques were observed under UV light (488 nm), a homogeneous population of mini-F recombinant HVT-miniF was obtained by 6 rounds of picking and plating on CEF cultures in a medium containing mycophenolic acid, xanthine, and hypoxanthine as described previously [34 (link)]. Genomic DNA extracted from these HVT-miniF CEF cultures was electroporated into E. coli DH10B competent cells (Invitrogen). Positive clones with chloramphenicol resistance were examined through RFLP with EcoR I and BamH I to select a clone of the HVT complete genome. The infectivity of HVT-BAC DNA was tested after transfection into CEFs using Lipofectamine 3000 (Invitrogen).
5
Generation of Bartha-K61 Infectious Clone
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A Bartha-K61 infectious clone was generated following the general technique of BAC [35 (link)]. Briefly, PRV Bartha-K61 DNA and pHA2-Puc19-H1B-H2B was co-transfected into ST cells, ensuring mini-F sequences insertion in the lieu of gC. After green plaques were observed under UV light (488 nm), plaque purification was carried out to obtain the homogeneous viruses, named B-mini-F. DNA of B-mini-F was transferred into E.coli DH10B competent cells (Invitrogen) by electroporation. Restriction fragment length polymorphism (RFLP) with Hind III and Sph I were determined to select positive clone of pB-mini-F, which was then electroporated into E.coli GS1783 for mutagenesis of Bartha-K61 genome [35 (link)].
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