Production of DMOS DNA Registers

The DMOS registers are made up of DNA sequences that have at least two dTdCdR sites (R = Purine, A or G). These sites have been proven to be effective binding sites based on the research conducted by Chari^{24 (link)} and synthesized by TWIST Bioscience. To facilitate the mass production of DMOS registers, we assembled the registers into the pBR322 plasmid (New England Biolabs) using the NEBuilder© HiFi Assembly Master Mix. We inserted the register into the plasmid by cleaving the pBR322 plasmid using FastDigest restriction enzymes Bsu15I and EcoRI.
Next, we transformed the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria and grew them under Carbenicillin antibiotic resistance. To extract the assembled plasmid, we used the Monarch Plasmid Miniprep Kit (NEB).

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Sadremomtaz A., Glass R.F., Guerrero J.E., LaJeunesse D.R., Josephs E.A, & Zadegan R. (2023). Digital data storage on DNA tape using CRISPR base editors. Nature Communications, 14, 6472.

Publication 2023

pBR322 plasmid NEBuilder© HiFi Assembly Master Mix Monarch Plasmid Miniprep Kit

Antibiotic resistance Bacteria Bacteria coli Binding sites Carbenicillin Dna sequences Ecori Escherichia Plasmid Purine Restriction enzymes

Corresponding Organization :

Other organizations : North Carolina Agricultural and Technical State University, University of North Carolina at Greensboro

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Variable analysis

independent variables

Presence of dTdCdR sites in the DNA sequences of the DMOS registers
Use of the pBR322 plasmid as the vector for assembly
Use of the NEBuilder© HiFi Assembly Master Mix for assembly
Use of the FastDigest restriction enzymes Bsu15I and EcoRI for plasmid cleavage
Transformation of the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria

dependent variables

Effectiveness of the dTdCdR sites as binding sites
Successful assembly of the DMOS registers into the pBR322 plasmid
Successful transformation of the modified plasmid into NEB 5-ɑ competent Escherichia coli bacteria
Extraction of the assembled plasmid using the Monarch Plasmid Miniprep Kit (NEB)

control variables

Use of the pBR322 plasmid as the vector for assembly
Use of the NEBuilder© HiFi Assembly Master Mix for assembly
Use of the FastDigest restriction enzymes Bsu15I and EcoRI for plasmid cleavage
Use of NEB 5-ɑ competent Escherichia coli bacteria for transformation
Use of Carbenicillin antibiotic resistance for bacterial growth

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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