All DNA sequences used in the development of the PLAN-LFA assay shown in Table 1 were purchased from Integrated DNA Technologies Inc. (Coralville, IA, USA). T4 DNA ligase, adenosine triphosphate (ATP), P1 nuclease (from P. citrinum), Phi29 DNA polymerase and deoxynucleotides were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). The supplied buffers were used for each of the ligation, amplification and nuclease protection steps of the assay. Nitrocellulose (FF120HP) and Whatman Grade 4 filter paper were purchased from GE Lifesciences (Pittsburgh, PA, USA). Streptavidin conjugated to Horseradish Peroxidase (Strep-HRP) was purchased from R&D Systems, Inc. (Minneapolis, MN). Pierce™ 1-Step Ultra TMB-Blotting Solution was purchased from Thermo Scientific (Waltham, MA). Anti-digoxigenin monoclonal antibody was purchased from Abcam, Inc (Cambridge, MA). StabilGuard® Immunoassay Stabilizer (BSA-free) was purchased from Surmodics, Inc. (Eden Prairie, MN). Trehalose dihydrate was purchased from EMD Millipore (Burlington, MA). Glycerol was purchased from Mallinkrodt (Staines-upon-Thames, United Kingdom).
Deoxynucleotides
Manufactured by New England Biolabs
Sourced in United States
Deoxynucleotides are the fundamental building blocks of DNA. They consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine, guanine, cytosine, or thymine. These molecules are essential for DNA synthesis and amplification in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (4 mentions)
Pierce 1 step ultra tmb blotting solution, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by New England Biolabs (1 mentions)
Nuclease p1, by New England Biolabs (1 mentions)
Trehalose dihydrate, by Merck Group (1 mentions)
Whatman grade 4 filter paper, by GE Healthcare (1 mentions)
Phi29 dna polymerase, by New England Biolabs (1 mentions)
E coli strain top10, by Thermo Fisher Scientific (1 mentions)
Q5 dna polymerase, by New England Biolabs (1 mentions)
Gentamicin sulfate, by Corning (1 mentions)
Ethidium homodimer, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Dna polymerase 1, by New England Biolabs (1 mentions)
N1 methyl pseudouridine, by TriLink (1 mentions)
3 o me m7g 5 ppp 5 g rna cap structure analog, by New England Biolabs (1 mentions)
Lb broth, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
Poly a polymerase, by New England Biolabs (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
4 protocols using deoxynucleotides
1
PLAN-LFA Assay Development Protocol
2
Plasmid Cloning and Transformation in E. coli
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Strains and plasmids used and constructed in this work are described in SI Appendix, Tables S1–S3 . E. coli strain TOP10 (Life Technologies) was used for cloning and amplification of plasmids. Plasmids were recovered using Econospin columns (Epoch Life Sciences) according to manufacturer’s instructions. Oligonucleotides were synthesized by the Stanford Protein and Nucleic Acid Facility. PCR was carried out using Q5 DNA Polymerase (New England Biolabs) unless otherwise specified, and all restriction enzymes, the T4 DNA ligase, and deoxynucleotides were purchased from New England Biolabs. Heterologous gene sequences were cloned from previously published plasmids, obtained from Addgene, or synthesized by Twist Bioscience. Cloning was exclusively performed using Gibson assembly (68 (link)) followed by transformation into E. coli or by gap-repair directly into yeast or direct genomic integration. Guide RNA (gRNA) integration plasmids were constructed from Addgene plasmid pCAS, which was a gift from Jamie Cate (Addgene plasmid # 60847) (69 (link)). pCAS was modified by Gibson assembly to create SpCas9 expression vector pCS3410, which was digested with PacI followed by Gibson assembly of each gRNA fragment. All gRNA sequences used in this work are listed in SI Appendix, Table S3 .
3
mRNA Transfection Protocol Using DMEM and Reagents
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Dulbecco’s Modified Eagle Medium (DMEM), Opti-MEM, 0.05% trypsin/0.53 mM EDTA and L-glutamine were all purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). Gentamicin Sulfate was from Corning (Corning, NY). All restriction enzymes, DNA polymerase I (Klenow), T4 DNA ligase, Deoxynucleotides, Poly(A) polymerase, 3′-O-Me m7G(5′)ppp(5′)G RNA cap structure analog, High Efficiency Competent E. Coli Cells [NEB 10-beta] and HiScribe™ T7 Quick High Yield RNA Synthesis Kit were from New England BioLabs (Ipswich, MA). Hi-Lo DNA Markers were obtained from Minnesota Molecular, Inc. (Minneapolis, MN). QIAprep spin miniprep kit was from Qiagen (Germantown, MD). LB Broth was purchased from Alfa Aesar (Ward Hill, MA). N1-Methylpseudouridine was from TriLink Biotechnologies (San Diego, CA). TransIT-mRNA lipid transfection kit was from Mirus Bio (Madison, WI). CellTiter 96® Aqueous One Solution Cell Proliferation Assay was from Promega (Madison, WI). Ethidium homodimer was purchased from Molecular Probes (Eugene, OR).
4
Purification and Characterization of StsA
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Materials and reagents were purchased from
Gold Biotechnology, Fisher Scientific, or Sigma-Aldrich unless otherwise
noted. Yeast extract and tryptone were purchased from Research Products
International. Molecular biology reagents for cloning (e.g., restriction
enzymes, Q5 polymerase, T4 DNA ligase, and deoxynucleotides) were
purchased from New England Biolabs. Oligonucleotide primers and gene
blocks were obtained from Integrated DNA Technologies. DNA spin columns
were purchased from Epoch Life Sciences. Sanger sequencing was performed
by the Roy J. Carver Biotechnology Center (University of Illinois
at Urbana-Champaign). Polymerase chain reactions were performed using
a Bio-Rad S1000 thermal cycler. Escherichia coli DH5α and BL21(DE3) strains were used for plasmid maintenance
and protein overexpression, respectively. Expressed StsA was purified
using an Agilent 1200 series HPLC fitted with a 10 × 250 mm C18
column (Macherey Nagel). Mass spectroscopy was performed using a Bruker
Daltonics UltrafleXtreme MALDI-TOF mass spectrometer and a ThermoFisher
Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate
100.
Gold Biotechnology, Fisher Scientific, or Sigma-Aldrich unless otherwise
noted. Yeast extract and tryptone were purchased from Research Products
International. Molecular biology reagents for cloning (e.g., restriction
enzymes, Q5 polymerase, T4 DNA ligase, and deoxynucleotides) were
purchased from New England Biolabs. Oligonucleotide primers and gene
blocks were obtained from Integrated DNA Technologies. DNA spin columns
were purchased from Epoch Life Sciences. Sanger sequencing was performed
by the Roy J. Carver Biotechnology Center (University of Illinois
at Urbana-Champaign). Polymerase chain reactions were performed using
a Bio-Rad S1000 thermal cycler. Escherichia coli DH5α and BL21(DE3) strains were used for plasmid maintenance
and protein overexpression, respectively. Expressed StsA was purified
using an Agilent 1200 series HPLC fitted with a 10 × 250 mm C18
column (Macherey Nagel). Mass spectroscopy was performed using a Bruker
Daltonics UltrafleXtreme MALDI-TOF mass spectrometer and a ThermoFisher
Scientific Orbitrap Fusion ESI-MS using an Advion TriVersa Nanomate
100.
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