Xl10 competent cells

Manufactured by Vazyme

Sourced in China

The XL10 competent cells are a high-efficiency bacterial transformation system designed for cloning and DNA manipulation. They are engineered to provide reliable and consistent transformation of a wide range of plasmid DNA.

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Lab products found in correlation

Nucleobond xtra midi plus, by Macherey-Nagel (1 mentions) Sali hf, by New England Biolabs (1 mentions) Random hexamer primer, by New England Biolabs (1 mentions) Ime199 dnap, by New England Biolabs (1 mentions) Phi29 dnap, by New England Biolabs (1 mentions) Puc19, by New England Biolabs (1 mentions)

2 protocols using xl10 competent cells

Genome Engineering of Plasmodium falciparum

Cited 1 time

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For enhancing transfection efficiency, the Cas9 endonuclease was integrated into the genome of wide type P. falciparum 3D7 strain for stable expression.³¹ (link) Specifically, C-terminal fragment (1219 bp) of PfP230p was amplified with primers P1/P2 (SI, Table S1) and cloned into pUF1-Cas9 plasmid at the restriction sites EcoRI and AvrII. Then the blasticidin cassette was replaced with yDHODH gene with primer P3/P4 (SI, Table S1) at the restriction sites NcoI and XbaI to obtain pUF1-DHODH-Cas9i plasmid.
Plasmids containing Pfkelch13 mutations were constructed based on the pL6cs-sgRNA plasmid (consisting of a sgRNA expression cassette and homology regions) via multiple cloning steps as previously described. Briefly, guide sequences (P5/P6) were cloned into pL6cs-sgRNA after annealing at the restriction sites AvrII and XhoI. Then two fragments of PF3D7_1343700 with desired mutations and corresponding donor DNA were amplified from genomic DNA 3D7 with the primers P7/P8, P9/P10, P11/P12, P13/P14 for Y493H, C580Y and homology regions, respectively (SI, Table S1). These fragments were cloned into the pL6cs-sgRNA plasmid using restriction sites AscI and AflII. The constructs were then transformed to XL10 competent cells (Vazyme Biotech Co., Ltd) for sequencing. Correctly sequenced plasmids were extracted by NucleoBond Xtra Midi Plus (Macherey-Nagel, Germany) for transfection.

Yu X., Wang C., Zhao Y., Tang J., Zhu M., Platon L., Culleton R., Zhu G., Ménard D., Zhang Q, & Cao J. (2022). Ring-stage growth arrest: Metabolic basis of artemisinin tolerance in Plasmodium falciparum. iScience, 26(1), 105725.

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Plasmid-based DNA Amplification and Fidelity

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Multiple displacement amplification assay was performed using the plasmid pUC19 (NEB, USA) as substrate. In brief, the pUC19 plasmid (NEB, USA) was mixed with random hexamer primers (NEB, USA) and incubated at 95 °C for 3 min and cooled to room temperature. The formed primed pUC19 DNA was added to the reaction mixture (25 μL, pH 7.5) contained 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA), 50 mM Tris -HCl, 10 mM (NH₄)₂SO₄, 10 mM MgCl₂, 500 μM dNTPs, 4 mM DTT, and incubated at 30 °C for 2 h. Electrophoresis and analysis were performed as described above.
The fidelity of IME199 DNAP was measured as previously described [15 (link)] with some modifications. In brief, IME199 DNAP or phi29 DNAP was incubated with 1 ng pUC19 (NEB, USA) at 30 °C for 8 h according to the above system. Amplification products were digested with restriction endonuclease SalI-HF (NEB, USA), heat inactivated, ligated, transformed into XL-10 competent cells (Vazyme, China), and plated onto Luria–Bertani solid plates containing ampicillin (50 μg/mL), X-Gal (0.8 mg) and IPTG (0.8 mg). Plates were incubated at 37 °C for 16 h and then the blue and white colonies were counted. In addition, the plasmids extracted from the white clones were sent to Ruibiotech (Beijing, China) for sequencing to identify the mutation site.

Han P., Fan H, & Tong Y. (2023). Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3). Microbial Cell Factories, 22, 217.

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