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Puregene yeast bact kit b

Manufactured by Qiagen
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The Puregene Yeast/Bact Kit B is a DNA extraction kit designed for the purification of genomic DNA from yeast and bacterial samples. The kit utilizes a simple and efficient protocol to isolate high-quality DNA for downstream applications.

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Lab products found in correlation

Hiseq 4000 system, by Illumina (2 mentions) Promethion platform, by Oxford Nanopore Technologies (2 mentions) Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Resuspension buffer, by Illumina (1 mentions) Nextera dna flex library prep kit, by Illumina (1 mentions) Qiaprep spin column, by Qiagen (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Tetracycline, by Merck Group (1 mentions) E coli top10f, by Thermo Fisher Scientific (1 mentions) Kanamycin, by Merck Group (1 mentions) Glycerol, by Avantor (1 mentions) Ampicillin, by Merck Group (1 mentions) Gentamicin, by Tokyo Chemical Industry (1 mentions) Qiaprep spin miniprep kit, by Qiagen (1 mentions) Trypticase soy broth (tsb), by BD (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Formedium (1 mentions)

8 protocols using puregene yeast bact kit b

1

Bacterial DNA Isolation Protocol

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An Escherichia coli K12 (MG1655) culture was grown overnight at 37 °C on an agar
plate. Next, a single colony was inoculated in 5 mL of autoclaved
LB medium (Invitrogen) with a sterile pipette and grown overnight
at 37 °C with continuous shaking. DNA extraction was performed
using the Qiagen Puregene Yeast/Bact Kit B according to the manufacturer’s
protocol. The concentration and purity of the DNA extracts were measured
using a Biodrop μlite UV–vis spectrophotometer.
D’Huys L., Vitale R., Ruppeka-Rupeika E., Goyvaerts V., Ruckebusch C, & Hofkens J. (2021). Assessing the Resolution of Methyltransferase-Mediated DNA Optical Mapping. ACS Omega, 6(33), 21276-21283.
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2

Mutation Rate Analysis of S. cerevisiae

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S. cerevisiae strains were grown in YPD (1% yeast extract, 2% Bacto Peptone and 2% dextrose) or in the appropriate synthetic dropout media (0.67% yeast nitrogen base without amino acids, 2% dextrose, and amino acid dropout mix at the concentration recommended by the manufacturer (US Biological) at 30 °C. All S. cerevisiae strains in this study were derived in the S288c strain background using standard gene deletion and pop-in, pop-out methods.
Mutator phenotypes were evaluated using the hom3-10 frameshift reversion assay. Mutation rates were determined by fluctuation analysis using a minimum of 2 independently derived strains and 14 or more independent cultures; comparisons of mutation rates were evaluated using 95% confidence intervals.
One independent Thr+ revertant was isolated per culture from fluctuation tests. Chromosomal DNA was isolated from each revertant using a Qiagen Puregene Yeast/Bact. Kit B and the hom3-10 region were amplified by PCR using the Primer 5′-AGT​TGT​TTG​TTG​ATG​ACT​GC and Primer 5′-TTC​AGA​AGC​TTC​TTC​TGG​AG and sequenced with the Primer 5′-CTT​TCC​TGG​TTC​AAG​CAT​TG using a commercial sequencing facility (Calil et al., 2021 (link)).
Miller A.K., Mao G., Knicely B.G., Daniels H.G., Rahal C., Putnam C.D., Kolodner R.D, & Goellner E.M. (2022). Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair. Frontiers in Cell and Developmental Biology, 10, 843121.
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3

Whole-Genome Sequencing of Bacteroides fragilis

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5 mL Tryptic soy broth (TSB) (BD BactoTM Cat # 6357914) + 5% Defibrinated Sheep blood (Remel, Cat # R54016, USA) liquid cultures from stock of clinical isolates were prepared and incubated under anaerobic conditions (90% N2, 5% CO2, 5% H2) at 37°C for 48 hrs. Then 1 mL of each isolate liquid culture was transferred to an Eppendorf tube and centrifuged at 13000 rpm for 60 seconds to collect bacteria. Total bacterial genome DNA of Bacteroides fragilis isolates was extracted, referring to steps in the instructions of the bacterial DNA extraction kit (Puregene yeast/Bact kit B, Qiagen, Cat#158567, USA). Extracted DNA samples were then quantified using a Qubit fluorometer (Thermo Fisher Scientific Inc., Cat# Q33238, USA) and library prepared using Illumina’s Nextera DNA Flex Library Prep kits (Cat# 20018705, USA) and following the manufacturer recommendation for the protocol. Only samples with a minimum of 100 ng DNA inputs were included. Pooled libraries were then quantified using the Qubit and diluted with Resuspension Buffer (Illumina, Cat# 20018705, USA) to 2 nM, then loaded on a NextSeq for sequencing with 50X sequencing depth.
Kordahi M.C., Stanaway I.B., Avril M., Chac D., Blanc M.P., Ross B., Diener C., Jain S., McCleary P., Parker A., Friedman V., Huang J., Burke W., Gibbons S.M., Willis A.D., Darveau R.P., Grady W.M., Ko C.W, & DePaolo R.W. (2021). Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer. Cell host & microbe, 29(10), 1589-1598.e6.
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4

Genome Sequencing and Analysis of Tigecycline-Resistant Isolate

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Total genomic DNA of the tigecycline-resistant isolate was extracted by Puregene Yeast/Bact Kit B (Qiagen, Maryland, US), and was sequenced by using Hiseq 4000 system (Illumina, San Diego, US) and PromethION platform (Nanopore, Oxford, UK). Hybrid assembly was performed by using Unicycler version 0.4.8 [28 (link)]. Antibiotic resistance genes were identified by ResFinder 3.2 [29 (link)] and CARD (https://card.mcmaster.ca/) with identity >80% and coverage >60%. Plasmid replicon typing was performed using PlasmidFinder v2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) with at least 95% identity and 60% coverage. Synteny analysis was performed using Easyfig [30 (link)]. Fragments >5 kb that were absent in at least one genome were detected by BLAST and were defined as genomic islands (GEIs) in this study as previously described [31 ]. Phylogenetic analysis with amino-acid sequences of Tet(X)s was performed by using the maximum likelihood method with default parameters by using Mega X Version 10.0.5 [32 (link)]. The amino acid sequences of Tet(X)s were submitted to ESPript 3 server [33 (link)] to perform the alignment and predict the secondary structure elements.
Cheng Y., Chen Y., Liu Y., Guo Y., Zhou Y., Xiao T., Zhang S., Xu H., Chen Y., Shan T., Xiao Y, & Zhou K. (2020). Identification of novel tetracycline resistance gene tet(X14) and its co-occurrence with tet(X2) in a tigecycline-resistant and colistin-resistant Empedobacter stercoris. Emerging Microbes & Infections, 9(1), 1843-1852.
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5

DNA Extraction and Molecular Cloning Protocols

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Chromosomal DNA was extracted from Bt cells with Puregene Yeast/Bact kit B (Qiagen, France). Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with QIAprep spin columns (Qiagen, France). PCR-amplified fragments and digested fragments separated on 0.8% agarose gels were purified with kits from Qiagen (France). Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations. Oligonucleotides (see Table S2 in the supplemental material) were synthesized by Sigma-Proligo (Paris, France) or Eurofins-MWG (Paris, France). PCRs were performed in a Applied Biosystems 2720 Thermak thermocycler (ABI). Nucleotide sequences of all constructs were determined by Beckman Coulter Genomics (Takeley, United Kingdom).
Verplaetse E., Slamti L., Gohar M, & Lereclus D. (2015). Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection. mBio, 6(3), e00138-15.
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6

Bacterial Strains and Plasmids Cultivation

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Bacterial strains and plasmids used in this study are listed in Table S3. E. coli and P. aeruginosa strains were grown in lysogeny broth (MP Biomedicals) at 37°C. Other Pseudomonas strains were cultured in Trypticase soy broth (TSB; BD Biosciences) at 30°C. All cultures underwent shaking at 200 rpm. Media were solidified with 1.5% agar (Invitrogen). Media were supplemented with filter-sterilized ampicillin (100 to 800 µg/ml; Sigma-Aldrich), isopropyl β-d-1-thiogalactopyranoside (IPTG; 25 µg/ml; ForMedium), kanamycin (50 µg/ml; Sigma-Aldrich), tetracycline (15 to 150 µg/ml; Sigma-Aldrich), or gentamicin (15 to 45 µg/ml; TCI Europe) when required. Bacterial stocks were stored in the appropriate medium at −80°C in 25% (vol/vol) glycerol (VWR).
Plasmids were propagated in E. coli TOP10F′ (Invitrogen). E. coli BL21(DE3) (Novagen) was used as a host for generating recombinant bacteriocins. Genomic DNA from Pseudomonas strains was collected with the Puregene Yeast/Bact kit B (Qiagen). Plasmid DNA was isolated using the QIAprep spin miniprep kit (Qiagen).
Ghequire M.G., Swings T., Michiels J., Buchanan S.K, & De Mot R. (2018). Hitting with a BAM: Selective Killing by Lectin-Like Bacteriocins. mBio, 9(2), e02138-17.
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7

Gram-negative Bacterial DNA Extraction

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DNA extraction was performed using the PureGene® Yeast/Bact kit B (Qiagen, Germany) following the manufacturer's instructions for extraction of DNA from Gram-negative bacteria and eluted in molecular grade water.
Hubbard A.T.M., & Roberts A. (2020). A novel hemA mutation is responsible for small colony variant phenotype in Escherichia coli.
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8

Genomic Analysis of Tigecycline-Resistant Bacteria

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Total genomic DNA of the tigecycline-resistant isolate was extracted by Puregene Yeast/Bact Kit B (Qiagen, Maryland, US), and was sequenced by using Hiseq 4000 system (Illumina, San Diego, US) and PromethION platform (Nanopore, Oxford, UK).
Hybrid assembly was performed by using Unicycler version 0.4.8 [28] . Antibiotic resistance genes were identified by ResFinder 3.2 with identity >90% and coverage >60% [29] . Synteny analysis was performed using Easyfig [30] .
Fragments >5 kb that were absent in at least one genome were detected by BLAST and were defined as genomic islands (GEIs) in this study as previously described [31] .
Phylogenetic analysis with amino-acid sequences of Tet(X)s was performed by using the maximum likelihood method with default parameters by using Mega X Version 10.0.5 [32] . The amino acid sequences of Tet(X)s were submitted to ESPript 3 server [33] to perform the alignment and predict the secondary structure elements.
Cheng Y., Chen Y., Liu Y., Guo Y., Zhou Y., Xiao T., Zhang S., Xu H., Chen Y., Shan T., Xiao Y., & Zhou K. (2020). Identification of novel tetracycline resistance genetet(X14) and its co-occurrence withtet(X2) in a tigecycline-resistant and colistin-resistantEmpedobacter stercoris.
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