C2566

Manufactured by New England Biolabs

Sourced in United States

C2566 is a thermal cycler instrument designed for DNA amplification and other temperature-controlled reactions. It features a compact design, a touch-screen interface, and supports standard PCR and qPCR protocols. The thermal cycler has a block capacity of 96 wells and can precisely control the temperature for efficient and reliable results.

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Lab products found in correlation

1 mm gap cuvette, by Bio-Rad (2 mentions) Escherichia coli dh5α, by New England Biolabs (1 mentions)

4 protocols using c2566

Bacterial Cultivation and Transformation

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EcNR2 18 , EcHB3 (Supplementary Table S6), C2566 (New England BioLabs, USA), and their derivatives were grown at 30°C (37°C for C2566) in Luria-Bertani (LB) broth (or agar) supplemented with appropriate antibiotics: ampicillin, 50 μg/ml; kanamycin, 30 μg/ml; or spectinomycin, 100 μg/ml.
IPTG was added to a final concentration of 1 mM. Electroporation was performed as follows: cells were grown until reaching an OD 600 of 0.7. Next, cells equivalent to 1 ml of culture medium were washed twice with 1 ml of ddH2O and resuspended in 50 μl of the appropriate product. The cells were then pulsed with 1.8 kV in a 1-mm-gap cuvette (Bio-Rad, USA).

Lim H., Jun S., Park M., Lim J., Jeong J., Lee J.H., & Bang D. (2020). Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system.

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Bacterial Cultivation and Transformation

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Lim H., Jun S., Park M., Lim J., Jeong J., Lee J.H, & Bang D. (2020). Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System. ACS synthetic biology, 9(5).

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Constructing Intein and TolC Libraries

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For cloning intein, the dummy control sequence in the pBR322-du1 plasmid was replaced by intein library sequences using NsiI and NcoI restriction sites. Gene kanR, which was constructed by LCR, was cloned into the pBR322 backbone plasmid using a one-step isothermal reaction (Gibson assembly35 (link)). Because the backbone vector is large, we divided it into two fragments with an 80 bp-overlap (Supplementary Fig. 3). The amplified gene insert and the two pBR322 backbone plasmid fragments were mixed in equal molar ratio to a final volume of 5 μl, and 15 μl assembly master mixture was added before incubation at 50 °C for 2 h. We constructed the tolC library using assembly PCR and cloned the inserts into the backbone vector by Gibson assembly. For cloning mcardinal, we first amplified gene products using both assembly PCR and LCR. We then cloned the amplicons into a pEGFP-C1 backbone plasmid using Gibson assembly and transformed into competent E. coli cells (C2566, NEB, USA).

Cho N., Hwang B., Yoon J.K., Park S., Lee J., Seo H.N., Lee J., Huh S., Chung J, & Bang D. (2015). De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries. Nature Communications, 6, 8351.

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Characterization of E. coli Strains

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Escherichia coli DH5α, BL21, and C2566 were purchased from New England Biolabs (USA). E. coli DH5α was used for DNA preparation and cloning. The cells were grown in lysogeny broth (LB) (Difco Laboratories Inc.,USA) with appropriate antibiotic supplementation (25 ug/ml kanamycin and 34 ug/ml chloramphenicol) for strain maintenance and plasmid construction in the different E. coli strains. Three E. coli strains (DH5α, BL21, C2566) were used for part characterization, which was conducted at 37°C on LB agar plates with optimum time-course for colony formation. The sequences of the DNA parts used in this study are presented in Table S1. The promoter and RBS were selected from the Registry (http://parts.igem.org) and the terminators were from [4 (link)]. For the DNA part preparation, the duplex oligosynthesis method of Macrogen (Korea) was employed.

Bak S.K., Seong W., Rha E., Lee H., Kim S.K., Kwon K.K., Kim H, & Lee S.G. (2022). Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology. Journal of Microbiology and Biotechnology, 32(8), 1026-1033.

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