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Mach1 chemically competent e coli

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The Mach1 chemically competent E. coli is a laboratory strain of Escherichia coli bacteria that has been genetically modified to be highly efficient at taking up and incorporating foreign DNA during a process called transformation. This product is designed for use in molecular biology and genetic engineering applications that require the introduction of plasmid DNA or other genetic material into bacterial cells.

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Lab products found in correlation

Kld enzyme mix, by New England Biolabs (2 mentions) Gblock gene fragment, by Integrated DNA Technologies (2 mentions) Phusion u green multiplex pcr master mix, by Thermo Fisher Scientific (2 mentions) Maxiprep, by Qiagen (2 mentions) Neb stable competent e coli, by New England Biolabs (2 mentions) Spcas9 hairpin u6 sgrna expression plasmid, by Addgene (2 mentions) Templiphi rolling circle amplification, by Thermo Fisher Scientific (2 mentions) 5 cbh aav abe npuc u6 sgrna, by Addgene (2 mentions) Aav abe npun, by Addgene (2 mentions) Bbsi plasmid digest, by New England Biolabs (2 mentions) User cloning, by New England Biolabs (2 mentions) Gibson assembly, by New England Biolabs (2 mentions)

5 protocols using mach1 chemically competent e coli

1

Mammalian Cell Expression Plasmid Construction

Cited 1 time
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All mammalian cell expression plasmids were constructed by USER cloning from gBlock gene fragments (Integrated DNA Technologies) with USER junctions sized between 14 and 20 nucleotides58 (link). Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. Mach1 chemically competent E. coli (ThermoFisher) cells were used.
Rees H.A., Yeh W.H, & Liu D.R. (2019). Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nature Communications, 10, 2212.
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2

Mammalian Cell Expression Plasmid Construction

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All mammalian cell expression plasmids were constructed by USER cloning from gBlock gene fragments (Integrated DNA Technologies), as previously described (30 (link)). Phusion U Green Multiplex PCR Master Mix (Thermo Fisher Scientific) was used for amplification of DNA. sgRNA plasmids were constructed by blunt-end ligation of a linear polymerase chain reaction (PCR) product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturer’s instruction. Mach1 chemically competent E. coli (Thermo Fisher Scientific) cells were used for plasmid construction.
Rees H.A., Wilson C., Doman J.L, & Liu D.R. (2019). Analysis and minimization of cellular RNA editing by DNA adenine base editors. Science Advances, 5(5), eaax5717.
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3

Plasmid Construction and AAV Cloning

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Base editor plasmids were constructed by replacing deaminase and Cas-protein domains of the p2T-CMV-ABE7.10-BlastR (Addgene 152989) plasmid by USER cloning (New England Biolabs)(50 (link)). Individual sgRNAs were cloned into the SpCas9-hairpin U6 sgRNA expression plasmid (Addgene 71485) using BbsI plasmid digest and Gibson assembly (New England Biolabs). Protospacer sequences and gene-specific primers used for amplification followed by HTS are listed in Supplementary Table 1. Constructs were transformed into Mach1 chemically competent E. coli (ThermoFisher) grown on LB agar plates and liquid cultures were grown in LB broth overnight at 37 °C with 100 μg/mL ampicillin. Individual colonies were validated by Templiphi rolling circle amplification (ThermoFisher) followed by Sanger sequencing. Verified plasmids were prepared by mini, midi, or maxiprep (Qiagen).
AAV vectors were cloned by Gibson assembly (NEB) using NEB Stable Competent E. coli (High Efficiency) to insert the sgRNA sequence and C-terminal base editor half of ABE8e-SpyMac into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half and a second U6-sgRNA cassette into v5 Cbh-AAV-ABE-NpuN (Addgene 137178)(74 (link)).
Arbab M., Matuszek Z., Kray K.M., Du A., Newby G.A., Blatnik A.J., Raguram A., Richter M.F., Zhao K.T., Levy J.M., Shen M.W., Arnold W.D., Wang D., Xie J., Gao G., Burghes A.H, & Liu D.R. (2023). Base editing rescue of spinal muscular atrophy in cells and in mice. Science (New York, N.Y.), 380(6642), eadg6518.
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4

Plasmid Construction and AAV Cloning

Cited 2 times
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Base editor plasmids were constructed by replacing deaminase and Cas-protein domains of the p2T-CMV-ABE7.10-BlastR (Addgene 152989) plasmid by USER cloning (New England Biolabs)(50 (link)). Individual sgRNAs were cloned into the SpCas9-hairpin U6 sgRNA expression plasmid (Addgene 71485) using BbsI plasmid digest and Gibson assembly (New England Biolabs). Protospacer sequences and gene-specific primers used for amplification followed by HTS are listed in Supplementary Table 1. Constructs were transformed into Mach1 chemically competent E. coli (ThermoFisher) grown on LB agar plates and liquid cultures were grown in LB broth overnight at 37 °C with 100 μg/mL ampicillin. Individual colonies were validated by Templiphi rolling circle amplification (ThermoFisher) followed by Sanger sequencing. Verified plasmids were prepared by mini, midi, or maxiprep (Qiagen).
AAV vectors were cloned by Gibson assembly (NEB) using NEB Stable Competent E. coli (High Efficiency) to insert the sgRNA sequence and C-terminal base editor half of ABE8e-SpyMac into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half and a second U6-sgRNA cassette into v5 Cbh-AAV-ABE-NpuN (Addgene 137178)(74 (link)).
Arbab M., Matuszek Z., Kray K.M., Du A., Newby G.A., Blatnik A.J., Raguram A., Richter M.F., Zhao K.T., Levy J.M., Shen M.W., Arnold W.D., Wang D., Xie J., Gao G., Burghes A.H, & Liu D.R. (2023). Base editing rescue of spinal muscular atrophy in cells and in mice. Science (New York, N.Y.), 380(6642), eadg6518.
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5

Customized Reporter Vector Construction

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The modification steps in the vector backbones were carried out using restriction enzyme cloning and T4 DNA Ligase as per manufacturer’s instructions. Propagation and transformation of Gateway cloning destination vectors was done in DB3.1 chemically competent E. coli (Thermo Fischer). To introduce responsive elements into pSBTET.reporter backbones, custom DNA oligos were designed using previously validated transcription factor binding sites such that upon annealing they generated PstI/XhoI overhangs (the TF binding site sequences used are provided in Supplemental Sequences, and complete vector sequences are available from Addgene). The annealed double-stranded oligos were then ligated into the vector backbone. Gateway reactions were carried out according to manufacturer’s instructions using the MultiSite Gateway Pro Plus Kit (Thermo Fischer). Entry Clones were created using Gateway BP Clonase II Enzymes, Expression clones were generated using Gateway LR Clonase II Plus. All Gateway reactions were transformed into MACH-1 chemically competent E. coli (Thermo Fischer).
Jäckel C., Nogueira M.S., Ehni N., Kraus C., Ranke J., Dohmann M., Noessner E, & Nelson P.J. (2016). A vector platform for the rapid and efficient engineering of stable complex transgenes. Scientific Reports, 6, 34365.
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