Conventional plasmid isolation and DNA manipulation techniques were used as described in Sambrook and Russell (44 ). Sanger sequencing was sourced to Elim Biopharm (California) and reaction composition were as per their instruction. Restriction enzymes and high-fidelity Phusion DNA polymerase were obtained from New England Biolabs (Ipswich, MA). Primers used in this study are listed in Supplemental Table 10 . For subcloning of PCR products pJET1.2 was used as an intermediate PCR cloning vector (ThermoScientific, Waltham, MA). GeneJET PCR purification, plasmid miniprep, and gel extraction kits (ThermoScientific, Waltham, MA) were routinely used. Ligation products were transformed by electroporation into E. coli EC100Dpir+ (Epicentre, Madison, WI).
Genejet pcr purification
Manufactured by Thermo Fisher Scientific
The GeneJET PCR Purification Kit is a product designed for the purification of PCR amplicons. It utilizes a silica-based membrane technology to efficiently capture and purify DNA fragments from PCR reactions, removing primers, nucleotides, polymerases, and other impurities.
Automatically generated - may contain errors
Lab products found in correlation
Gel extraction kit, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
E coli ec100dpir, by Illumina (2 mentions)
Pjet1, by Thermo Fisher Scientific (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep, by Thermo Fisher Scientific (1 mentions)
H3bo3, by Merck Group (1 mentions)
Mgso4 7h2o, by Merck Group (1 mentions)
Na2edta, by Merck Group (1 mentions)
Rnasezap, by Merck Group (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
5 protocols using genejet pcr purification
1
Molecular Cloning Techniques for DNA Manipulation
2
Molecular Cloning Techniques for DNA Manipulation
3
Quantification of Episomal DNA by Exonuclease V Assay
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Episomal DNA was measured using an exonuclease V assay described by the Sapp lab (26 (link)). Total DNA was isolated from cells using phenol-chloroform followed by precipitation with ethanol. DNA was then quantitated using Qubit 4 fluorometer with the BR DNA assay kit (Invitrogen), and 1 μg of DNA was digested using the exonuclease V. DNA was then purified using GeneJet PCR purification (Thermo Fisher) and amplified with Roche light cycler 480 using primers specific to E7. The forward primer was 5′-ATGAGCAATTACCCGACAGCTCAGA , and the reverse primer was 5′-AGACTTACACTGACAACAAAAGGTAACGAT .
4
Comprehensive Cyanobacterial DNA Extraction
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The chemicals used in this study were purchased from Fisher Scientific (NaCl, Tris base), Amresco (KCl, ampicillin disodium salt, and kanamycin monosulfate, bacteriological agar), and Sigma-Aldrich (MgSO4·7H2O, CuSO4·5H2O, Na2EDTA, H3BO3, CaCl2·2H2O, KH2PO4, NaNO3, vitamin B12, FeCl3·6H2O, MnCl2·4H2O, ZnCl2, CoCl2·6H2O, RNaseZAP). Primers were synthesized by Sigma-Aldrich. GenElute Bacterial Genomic DNA kit from Sigma-Aldrich was used to isolate cyanobacterial genomic DNA. Other kits utilized in the molecular biological methods were GeneJET Plasmid Miniprep, GeneJET PCR purification, and GeneJET Gel Extraction DNA Recovery kits, FD restriction enzymes, DNA polymerases, and ligases, all purchased from Thermo-Scientific. Suppliers of all other materials used in this study are described below.
5
In-Vitro Synthesis of CRISPR sgRNA
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Using two primers, sgRNA_For and sgRNA_PSP1_Rev (Supplementary Table 1 ), the DNA sequence encoding the T7 RNA polymerase promoter and sgRNA was amplified by PCR from pHelper_ShCAST_sgRNA vector (Addgene, #127921). The DNA template was then subjected to GeneJet PCR purification (ThermoScientific). sgRNA was produced by in vitro transcription using the HiScribe T7 High Yield RNA synthesis kit (NEB) with the PCR amplified DNA sequence as the template. Purified sgRNA was aliquoted and stored at −20 °C.
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