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monarch pcr dna cleanup spin columns  (New England Biolabs)


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    New England Biolabs monarch pcr dna cleanup spin columns
    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by <t>PCR</t> from either BsmBI-domesticated T7 genomic <t>DNA</t> or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.
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    Images

    1) Product Images from "Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water"

    Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.5c00665

    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.
    Figure Legend Snippet: Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

    Techniques Used: Generated, Transformation Assay, Electroporation, Isolation, Variant Assay, Luciferase, Infection, Lysis, Bacteria

    Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.
    Figure Legend Snippet: Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

    Techniques Used: Modification, Amplification, Size Selection, Nucleic Acid Purification, Biomarker Discovery, Nucleic Acid Electrophoresis, Electroporation, Isolation, Sequencing

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    Purification:

    Article Title: Redefining rock doves, Columba livia , using historical whole genome sequences
    Article Snippet: .. Tissue for each sample was was digested overnight in 0.3ml of the aforementioned digestion buffer., which was purified directly over Monarch DNA Cleanup Columns (5 μg) (New England Biolabs) using a 10:1 ratio of binding buffer (a modified version of Qiagen’s PB buffer) to digestive mixture, repeated 3 times. .. PE buffer (Qiagen) was then used to wash the sample, followed by a dry spin to remove any remaining residue, and finalised with two consecutive elutions to increase DNA yield, each of 15 μL of EB (Qiagen) buffer incubated at 37 C for 10 minutes.

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance
    Article Snippet: We combined 100ng of linearized vector, 1.08 μL barcode oligo insert (100 nM in nuclease-free water), 10 μL Gibson assembly master mix (NEB E2611) and nuclease free water to a final volume of 20 μL then incubated the reaction at 50oC for 1 hour. .. We next column purified the assembled plasmid using Monarch DNA cleanup columns (NEB) according to the manufacturer’s protocol then electroporated 2 μL of the column purified plasmid into Endura Electrocompetent E. coli cells (Lucigen) using a GenePulserXCell (Biorad) with the following settings: 25msec pulse length, 10 μF capacitance, 600Ω resistance, and 1800V voltage. ..

    Article Title: De novo, high-quality assembly and annotation of the halophyte grass Aeluropus littoralis draft genome and identification of A20/AN1 zinc finger protein family
    Article Snippet: .. Following the purification of the library using PB binding buffer (Qiagen, Hilden, Germany) and Monarch DNA Cleanup Columns (New England Biolabs, Massachusetts, USA), the library was eluted in 40 μL of buffer EB (Qiagen, Hilden, Germany). .. Post-PCR amplification, as an extra purification step, was performed via the AMpure XP system (Agentcourt, Beckman Counter, Indianapolis, USA) with a 1.8 × bead: library ratio to remove any persisting primer dimers or other molecules with a fragment size of <100 bp.

    Article Title: De novo, high-quality assembly and annotation of the halophyte grass Aeluropus littoralis draft genome and identification of A20/AN1 zinc finger protein family.
    Article Snippet: .. Following the purification of the library using PB binding buffer (Qiagen, Hilden, Germany) and Monarch DNA Cleanup Columns (New England Biolabs, Massachusetts, USA), the library was eluted in 40 μL of buffer EB (Qiagen, Hilden, Germany). .. Post-PCR amplification, as an extra purification step, was performed via the AMpure XP system (Agentcourt, Beckman Counter, Indianapolis, USA) with a 1.8 × bead: library ratio to remove any persisting primer dimers or other molecules with a fragment size of <100 bp.

    Article Title: A genome-scale single cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines
    Article Snippet: The mScarlet sequence was amplified by PCR from plasmid pmScarlet_C1 (Addgene 85042) using primers #1016 and #1012 ( Supplementary Table STM-1 ). .. All products were purified with Monarch DNA Cleanup Columns (NEB). .. T2A sequence was ordered as a gBlock from IDT.

    Binding Assay:

    Article Title: Redefining rock doves, Columba livia , using historical whole genome sequences
    Article Snippet: .. Tissue for each sample was was digested overnight in 0.3ml of the aforementioned digestion buffer., which was purified directly over Monarch DNA Cleanup Columns (5 μg) (New England Biolabs) using a 10:1 ratio of binding buffer (a modified version of Qiagen’s PB buffer) to digestive mixture, repeated 3 times. .. PE buffer (Qiagen) was then used to wash the sample, followed by a dry spin to remove any remaining residue, and finalised with two consecutive elutions to increase DNA yield, each of 15 μL of EB (Qiagen) buffer incubated at 37 C for 10 minutes.

    Article Title: De novo, high-quality assembly and annotation of the halophyte grass Aeluropus littoralis draft genome and identification of A20/AN1 zinc finger protein family
    Article Snippet: .. Following the purification of the library using PB binding buffer (Qiagen, Hilden, Germany) and Monarch DNA Cleanup Columns (New England Biolabs, Massachusetts, USA), the library was eluted in 40 μL of buffer EB (Qiagen, Hilden, Germany). .. Post-PCR amplification, as an extra purification step, was performed via the AMpure XP system (Agentcourt, Beckman Counter, Indianapolis, USA) with a 1.8 × bead: library ratio to remove any persisting primer dimers or other molecules with a fragment size of <100 bp.

    Article Title: Road to Extinction? Past and Present Population Structure and Genomic Diversity in the Koala
    Article Snippet: .. The digest was centrifuged, and the supernatant was collected and mixed with 8 × of a binding buffer as detailed in then centrifuged through Monarch DNA Cleanup Columns (5 μg) (New England Biolabs Inc. Beverly, MA, USA). .. DNA bound to the columns was washed with 800 μL buffer PE (Qiagen, Hilden, Germany), then eluted in 25 μL buffer EB (Qiagen).

    Article Title: De novo, high-quality assembly and annotation of the halophyte grass Aeluropus littoralis draft genome and identification of A20/AN1 zinc finger protein family.
    Article Snippet: .. Following the purification of the library using PB binding buffer (Qiagen, Hilden, Germany) and Monarch DNA Cleanup Columns (New England Biolabs, Massachusetts, USA), the library was eluted in 40 μL of buffer EB (Qiagen, Hilden, Germany). .. Post-PCR amplification, as an extra purification step, was performed via the AMpure XP system (Agentcourt, Beckman Counter, Indianapolis, USA) with a 1.8 × bead: library ratio to remove any persisting primer dimers or other molecules with a fragment size of <100 bp.

    Modification:

    Article Title: Redefining rock doves, Columba livia , using historical whole genome sequences
    Article Snippet: .. Tissue for each sample was was digested overnight in 0.3ml of the aforementioned digestion buffer., which was purified directly over Monarch DNA Cleanup Columns (5 μg) (New England Biolabs) using a 10:1 ratio of binding buffer (a modified version of Qiagen’s PB buffer) to digestive mixture, repeated 3 times. .. PE buffer (Qiagen) was then used to wash the sample, followed by a dry spin to remove any remaining residue, and finalised with two consecutive elutions to increase DNA yield, each of 15 μL of EB (Qiagen) buffer incubated at 37 C for 10 minutes.

    Plasmid Preparation:

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance
    Article Snippet: We combined 100ng of linearized vector, 1.08 μL barcode oligo insert (100 nM in nuclease-free water), 10 μL Gibson assembly master mix (NEB E2611) and nuclease free water to a final volume of 20 μL then incubated the reaction at 50oC for 1 hour. .. We next column purified the assembled plasmid using Monarch DNA cleanup columns (NEB) according to the manufacturer’s protocol then electroporated 2 μL of the column purified plasmid into Endura Electrocompetent E. coli cells (Lucigen) using a GenePulserXCell (Biorad) with the following settings: 25msec pulse length, 10 μF capacitance, 600Ω resistance, and 1800V voltage. ..



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    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by <t>PCR</t> from either BsmBI-domesticated T7 genomic <t>DNA</t> or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.
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    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by <t>PCR</t> from either BsmBI-domesticated T7 genomic <t>DNA</t> or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.
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    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by <t>PCR</t> from either BsmBI-domesticated T7 genomic <t>DNA</t> or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.
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    Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

    Journal: ACS Synthetic Biology

    Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

    doi: 10.1021/acssynbio.5c00665

    Figure Lengend Snippet: Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

    Article Snippet: Initial purification of the PCR amplicons was conducted using Monarch PCR & DNA Cleanup spin columns (New England Biolabs, Ipswitch, MA).

    Techniques: Generated, Transformation Assay, Electroporation, Isolation, Variant Assay, Luciferase, Infection, Lysis, Bacteria

    Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

    Journal: ACS Synthetic Biology

    Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

    doi: 10.1021/acssynbio.5c00665

    Figure Lengend Snippet: Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

    Article Snippet: Initial purification of the PCR amplicons was conducted using Monarch PCR & DNA Cleanup spin columns (New England Biolabs, Ipswitch, MA).

    Techniques: Modification, Amplification, Size Selection, Nucleic Acid Purification, Biomarker Discovery, Nucleic Acid Electrophoresis, Electroporation, Isolation, Sequencing