The genomic DNA of the phage was extracted using the Norgen Biotek Phage DNA Isolation Kit (Norgen Biotek Corp. Thorold, ON, Canada). Briefly, phage genomic DNA was obtained through 4 steps of propagating the phage in liquid cultures and preparing the phage supernatant containing 109 PFU/mL, lysate preparation, sample binding to column, column washing, and DNA elution. Genomic DNA sequencing was performed using the Illumina Novaseq platform (Shanghai Personalbio Technology Co., Ltd. Shanghai, China). The standard Illumina TruSeq Nano DNA LT library preparations (Illumina TruSeq DNA Sample Preparation Guide) were used to construct the required online genome libraries. Genemarks [24 (link)] software was used to predict the protein-coding genes of the genome, and the sequence alignment of the coding genes was completed by Diamond software [25 (link)]. The putative protein function of the ORFs was annotated using the NR database. Diamond BLASTp was used to compare the protein sequences encoded by genes with the protein sequences in the database, to identify the function of the presumed proteins. Non-coding RNAs in the genome were predicted by comparing the ncRNA annotation database http://rfam.xfam.org/ (accessed on 13 August 2021) [26 (link)]. Phylogenetic analysis was conducted using MEGA (version X _10.2.6, Auckland, New Zealand).
Phage dna isolation kit
Manufactured by Norgen Biotek
Sourced in Canada, United States
The Phage DNA Isolation Kit is a laboratory equipment designed to extract and purify DNA from bacteriophages, which are viruses that infect bacteria. The kit provides a streamlined process for isolating phage DNA from various sample types, allowing researchers to study the genetic composition and characteristics of these viruses.
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Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Miseq sequencing platform, by Illumina (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Nextseq 550, by Illumina (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Rnase a, by Merck Group (2 mentions)
Purelink microbiome dna purification kit, by Thermo Fisher Scientific (2 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Bcl convert, by Illumina (2 mentions)
Dna prep kit, by Illumina (2 mentions)
Hiseq 4000 instrument, by Illumina (2 mentions)
Truseq dna pcr free library preparation kit, by Illumina (2 mentions)
Dneasy powersoil pro kit, by Qiagen (2 mentions)
Qiacube, by Qiagen (2 mentions)
81 protocols using phage dna isolation kit
1
Phage Genomic DNA Extraction and Sequencing
2
Bacteriophage Genome Sequencing and Annotation
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Bacteriophage genomes were purified using the Norgen Biotek Phage DNA Isolation Kit and sequenced at the former Microbial Genome Sequencing Center (MiGS) using Illumina Technology. Genome assembly and downstream analyses were performed using Geneious Prime 2021.0.1 following standard procedures in the field73 (link). Phage genomes were annotated using Pharokka v1.3.074 (link) followed by manual curation. Coding sequences (CDS) were predicted with PHANOTATE v1.5.175 (link) and tRNAs were predicted with tRNAscan-SE v2.0.1176 (link).
3
Construction and Analysis of Fluorescent Phage SPP1
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For fluorescence microscopy, bacterial cells (0.5 mL, OD 600 0.5) were centrifuged and suspended in 50 mL of MMB. For time lapse microscopy, bacteria were placed over 1% MMB agarose pad and incubated in a temperature controlled chamber (Pecon-Zeiss) at 37 C. Samples were photographed using Axio Observer Z1 (Zeiss), equipped with CoolSnap HQII camera (Photometrics, Roper Scientific). System control and image processing were performed using MetaMorph 7.4 software (Molecular Devices).
Construction of Fluorescent Phages SPP1 DNA was extracted using Phage DNA Isolation Kit (Norgen Biotek Corp). SPP1 was assembled from 4 DNA fragments (10-12 kb), lacking gp51, amplified using KAPA HiFi HotStart PCR Kit (Kapa Biosystems). gp51 was added as gp51-yfp fragment and SPP1 DNA was assembled in vitro using Gibson assembly master mix (New England Bio labs), and transformed into PY79 using a standard procedure (Harwood and Cutting, 1990) . The constructed phage was subjected to whole genome sequence analysis (Illumina), to confirm the correct insertion of the yfp and the assembly of the fragments (yfp was inserted at position 21654). Cells were plated on MMB plates, and plaques were tested for their fluorescence and infection capability.
Construction of Fluorescent Phages SPP1 DNA was extracted using Phage DNA Isolation Kit (Norgen Biotek Corp). SPP1 was assembled from 4 DNA fragments (10-12 kb), lacking gp51, amplified using KAPA HiFi HotStart PCR Kit (Kapa Biosystems). gp51 was added as gp51-yfp fragment and SPP1 DNA was assembled in vitro using Gibson assembly master mix (New England Bio labs), and transformed into PY79 using a standard procedure (Harwood and Cutting, 1990) . The constructed phage was subjected to whole genome sequence analysis (Illumina), to confirm the correct insertion of the yfp and the assembly of the fragments (yfp was inserted at position 21654). Cells were plated on MMB plates, and plaques were tested for their fluorescence and infection capability.
4
Phage DNA Extraction Protocol
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DNA was extracted from CsCl purified high-titre stocks of phage using phage DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. The purity and the concentration of the DNA were determined using spectrophotometer (Invitrogen Qubit).
5
Viral DNA Extraction and qPCR Analysis
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Viral preparations consisted of non-chromosomal DNA (RQ1, RNase-Free DNase, Promega) prepared from 1 ml of cells of the indicated strains. Overnight cultures were back-diluted 1:100 and grown for 90 min before being divided into 3 equal volumes and exposed to treatments as specified. Cultures were grown for an additional 5 h before collection of cell-free culture fluids (Corning SpinX). qPCR reactions were performed as described above for RT–qPCR assays. Next, 1 µl of purified non-chromosomal DNA was used for each qPCR reaction. Data were analysed by normalizing the CT values of samples treated with ciprofloxacin or aTc to the CT values of samples treated with water using a primer set to the indicated phage. Viral preparations for whole-genome sequencing (SeqCenter) were prepared exactly as described for qPCR, except by column purification (Phage DNA Isolation Kit, Norgen Biotek).
6
Phage DNA Isolation and Purification
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DNase I (Sigma-Aldrich, Germany) and RNase A (Sigma-Aldrich) treatment was performed to remove any exogenous host genomic DNA and RNA from purified phage particles as described previously70 (link). The DNA was isolated with a Phage DNA Isolation Kit (Norgen Biotek Corporation, Canada) according to the manufacturer’s recommendations. DNA from the viral particles for sequencing was isolated by phenol-chloroform extraction62 (link). The concentration and purity of the phage DNA was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).
7
Whole-Genome Sequencing and Annotation of KFS-EC3 Phage
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The genomic DNA of KFS-EC3 was extracted and purified using a Phage DNA Isolation Kit (Norgen Biotek Corp. Thorold, ON, Canada). Whole-genome sequencing of the purified DNA was performed (Macrogen Inc., Seoul, Korea) using the paired-end Miseq sequencing platform (Illumina Inc., San Diego, CA, USA). The raw reads were trimmed using Trimmomatic [28 (link)] to eliminate the low-quality reads and adapter sequences. The de novo assembly of the qualified sequences was performed by various k-mer using the SPAdes genome assembler (Illumina). The open reading frames (ORFs) of the assembled sequence were predicted and annotated using the Rapid Annotations using Subsystems Technology (RAST) server [29 (link)] and BLASTP. The potential tRNA genes in the genome sequence were predicted using ARAGORN [30 (link)] and RNAmmer (version 1.2) [31 (link)] in the rapid prokaryotic genome annotation pipeline (Prokka) [32 (link)]. The complete genome sequence of KFS-EC3 was deposited in the GenBank database, under the nucleotide sequence accession number MZ065353. The novelty of phage was assessed based on its similarity to other most closely related phages from the NCBI BLASTN database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch , 5 May 2021).
8
Phage DNA Extraction and Sequencing
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DNA extraction was performed by incubating 1 mL of purified phage lysate (1010 PFU/mL) with DNase I (final concentration 1 ug/mL) and RNase A (final concentration 30 ug/mL) to remove host nucleic acid contamination. After 30 min of incubation at room temperature, the mixture was heated at 75 °C for 5 min to inactivate DNase I. Then, the mixture was treated with 4 µL of proteinase K (20 mg/mL) and lysis buffer B (Phage DNA isolation kit) for 1 h at 56 °C. DNA was further extracted using the Phage DNA isolation kit (Norgen, Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions. The extracted phage DNA was subsequently sequenced.
9
Phage Genome Sequencing and Annotation
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Phage DNA was extracted using the Norgen Biotek Corp phage DNA isolation kit as per the manufacturer’s instructions (Norgen Biotek Corp., Thorold, Ontario, Canada). Phage genome sequencing was performed by GenProbio at the University of Parma, Italy. Genomes were sequenced with Illumina MiSeq Sequencing System and assembled with MIRA v4.0.2. De novo sequence assemblies and automated gene calling was performed using the MEGAnnotator pipeline [24 (link)] and assessed for predicted transfer RNA genes via tRNAscan-SE v1.2.1 [25 (link)]. Predicted open reading frames (ORFs) were determined via Prodigal v2.6 [26 (link)]. A BLASTP [27 (link)] analysis was performed to assign functional annotations to the predicted ORFs (https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The proposed functional annotations were further investigated by performing structural homology searches via HHpred [28 (link)] and querying the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). The annotated genomes were manually inspected, edited and finalized using the Artemis visualization tool [29 (link)].
10
Phage DNA Isolation and Storage
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Ec_MI-02 was propagated overnight in LBB to reach a titer of 108–109 pfu/mL as described previously [62 (link)]. One mL of the suspension was used for DNA isolation using a phage DNA isolation kit (Norgen Biotek Corp, Thorold, ON, Canada), following the manufacturer’s protocol. The extracted DNA was stored at −20 °C for 48 h.
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