Antibiotics (Gold Biotechnology) were used at the following working concentrations: carbenicillin 50 μg/mL, spectinomycin 50 μg/mL, chloramphenicol 25 μg/mL, kanamycin 50 μg/mL, tetracycline 10 μg/mL, streptomycin 50 μg/mL. Nuclease-free water (Qiagen) was used for PCR reactions and cloning. For all other experiments, water was purified using a MilliQ purification system (Millipore). Unless otherwise noted, Phusion U Hot Start or Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) were used for all PCRs. Unless otherwise noted, plasmids and SPs were cloned by USER assembly26 (link). Genes were obtained as synthesized gene fragments from Twist Bioscience. Plasmids were cloned and amplified using either Mach1 (Thermo Fisher Scientific) or Turbo (New England BioLabs) cells. Plasmid or SP DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) prior to Sanger sequencing. Strain S206025 (link) was used in all luciferase, phage propagation, and plaque assays, and in all PACE experiments. A description of plasmids, SP, primers, and protospacer sequences used in this work is provided in Table S4 .
Phusion u hot start
Manufactured by Thermo Fisher Scientific
Sourced in France
Phusion U Hot Start is a high-fidelity DNA polymerase designed for reliable and efficient PCR amplification. It provides robust performance and accuracy, ensuring consistent and accurate DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Milli q purification system, by Merck Group (2 mentions)
Illustra templiphi 100 amplification kit, by GE Healthcare (2 mentions)
Mach1, by Thermo Fisher Scientific (2 mentions)
Phusion hot start 2 dna polymerase, by Thermo Fisher Scientific (2 mentions)
Nuclease free water, by Qiagen (2 mentions)
Pyrogold sqa reagent kit, by Qiagen (2 mentions)
Pyromark cpg software, by Qiagen (2 mentions)
Hotstar taq polymerase, by Qiagen (2 mentions)
Sybr gold nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Agencourt dnadvance genomic dna isolation kit, by Beckman Coulter (1 mentions)
Hotstartaq buffer, by Qiagen (1 mentions)
5 protocols using phusion u hot start
1
Antibiotic and Nuclease-free Reagents for Molecular Biology
2
DNA Extraction and Sequencing Protocol
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Genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer’s instructions. First DNA amplification was performed by quantitative PCR with Phusion U Hot Start and SYBR Gold Nucleic Acid Stain (Thermo Fisher) to the top of the linear range. PCR products were purified using RapidTips (Diffinity Genomics). The second PCR was performed to attach Indexing Adapters (Illumina). The products were gel-purified and quantified using the KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced using a single-end read from 200–250 bases (depending on the amplicon size) on the MiSeq (Illumina) according to the manufacturer’s instructions.
3
Quantitative DNA Methylation Analysis by Pyrosequencing
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Quantitative DNA methylation analysis for validation was performed by pyrosequencing of bisulfite-treated DNA [Citation43] . Six regions of interest for validation were amplified using 30 ng of bisulfite-treated human genomic DNA and 5 to 7.5 pmol of forward and reverse primer, one of them being biotinylated. Sequences for oligonucleotides for PCR amplification and pyrosequencing are shown in Supplementary Table 1. Reaction conditions were either 1X HotStar Taq buffer supplemented with 1.6 mM MgCl 2 , 100 μM dNTPs, 5 pM of each primer and 2.0 U HotStar Taq polymerase (Qiagen, Courtaboeuf, France) in a 25 μl volume or 1X Phusion U Hotstart (Thermo Fisher Scientific, Les Ulis, France) with 5 pM of each primer. The PCR program consisted of a denaturing step of 15 min at 95°C followed by 50 cycles of 30 s at 95°C, 30 s at the respective annealing temperature and 20 s at 72°C, with a final extension of 5 min at 72°C. 10 μl of PCR product were rendered single-stranded as previously described [Citation43] and 4 pmol of the respective sequencing primer were used for analysis. Quantitative DNA methylation analysis was carried out on a PSQ 96MD system with the PyroGold SQA Reagent Kit (Qiagen) and results were analyzed using the PyroMark CpG software (V.1.0.11.14, Qiagen).
4
Antibiotic and Nuclease-free Reagents for Molecular Biology
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Antibiotics (Gold Biotechnology) were used at the following working concentrations: carbenicillin 50 μg/mL, spectinomycin 50 μg/mL, chloramphenicol 25 μg/mL, kanamycin 50 μg/mL, tetracycline 10 μg/mL, streptomycin 50 μg/mL. Nuclease-free water (Qiagen) was used for PCR reactions and cloning. For all other experiments, water was purified using a MilliQ purification system (Millipore). Unless otherwise noted, Phusion U Hot Start or Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) were used for all PCRs. Unless otherwise noted, plasmids and SPs were cloned by USER assembly26 (link). Genes were obtained as synthesized gene fragments from Twist Bioscience. Plasmids were cloned and amplified using either Mach1 (Thermo Fisher Scientific) or Turbo (New England BioLabs) cells. Plasmid or SP DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) prior to Sanger sequencing. Strain S206025 (link) was used in all luciferase, phage propagation, and plaque assays, and in all PACE experiments. A description of plasmids, SP, primers, and protospacer sequences used in this work is provided in Table S4 .
5
Pyrosequencing for DNA Methylation Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative DNA methylation analysis for validation was performed by pyrosequencing of bisulfite-treated DNA [41] . Six regions of interest for validation were amplified using 30 ng of bisulfite-treated human genomic DNA and 5 to 7.5 pmol of forward and reverse primer, one of them being biotinylated. Sequences for oligonucleotides for PCR amplification and pyrosequencing are shown in Supplementary Table 1. Reaction conditions were either 1x HotStar Taq buffer supplemented with 1.6 mM MgCl 2 , 100 µM dNTPs, 5 pM of each primer and 2.0 U HotStar Taq polymerase (Qiagen) in a 25 µl volume or 1X Phusion U Hotstart (Thermo Fisher Scientific) with 5 pM of each primer. The PCR program consisted of a denaturing step of 15 min at 95°C followed by 50 cycles of 30 s at 95°C, 30 s at the respective annealing temperature and 20 s at 72°C, with a final extension of 5 min at 72°C. 10 µl of PCR product were rendered single-stranded as previously described [41] and 4 pmol of the respective sequencing primer were used for analysis. Quantitative DNA methylation analysis was carried out on a PSQ 96MD system with the PyroGold SQA Reagent Kit (Qiagen) and results were analyzed using the PyroMark CpG software (V.1.0.11.14, Qiagen).
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