Chromatin was fixed with 3% paraformaldehyde followed by further cross-linking with 10 mM dimethyl adipimidate (DMA). Crosslinked chromatin was sheared into 300–500 bp DNA fragments by Bioruptor (Diagenode). Cut14-Pk and Rad21-Myc proteins were immunoprecipitated using mouse monoclonal anti-PK (SV5-Pk1, Serotec) or mouse monoclonal anti-Myc (9E10, Clontech), and protein G-coupled Dynabeads (Life Technologies). Immunoprecipitated chromatin was subjected to the ChIA-PET procedure20 . ChIP DNA was split into an equal amount and separately ligated to either A or B linkers, and mixed ligation products were further subjected to proximity ligation. The ligation product was digested by EcoP15I and purified using streptavidin-coupled magnetic beads (Invitrogen). Illumina sequencing adaptors were added to DNA fragments, which were then subjected to PCR amplification (Phusion PCR Master Mix; New England Biolabs). This PCR was performed in a total of 32 separate tubes for each ChIA-PET library. Assembled PCR products were sequenced on the Illumina HiSeq 2000 platform to obtain 100 bp single end reads.
Phusion pcr master mix
Manufactured by New England Biolabs
Sourced in United States, China
Phusion PCR Master Mix is a high-fidelity DNA polymerase specifically designed for accurate DNA amplification. It provides a robust and reliable solution for a wide range of PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sv5 pk1, by Bio-Rad (2 mentions)
Mouse monoclonal anti myc 9e10, by Takara Bio (2 mentions)
Protein g coupled dynabeads, by Thermo Fisher Scientific (2 mentions)
Streptavidin coupled magnetic beads, by Thermo Fisher Scientific (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Milli q plus 185 water purification system, by Merck Group (1 mentions)
Poly lactic co glycolic acid plga, by Jinan Daigang Biomaterial (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
4 protocols using phusion pcr master mix
1
Chromatin Crosslinking and ChIA-PET
2
Generation of α-Amylase Mutant Using OE-PCR
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The α-amylase mutant gene was generated using the Overlap Extension PCR (OE-PCR) method. The external forward primer contained an EcoRI restriction site, and the external reverse primer contained an XhoI restriction site. The change in nucleotide sequence was introduced by incorporating nucleotide changes into the overlapping oligo primers, as shown in Supplemental file . During the cloning process, the native signal peptide DNA sequence was excluded, as the pET-22b(+ ) contains a pelB leading sequence. Phusion® PCR Master Mix (NEB, Ipswich, Massachusetts, USA) was used to perform the PCR. PCR reaction mixtures containing 25 ng ASKA DNA template, 25 μl 2× PhusionTM Flash PCR Master Mix, and 0.5 μM each primer were added to a final volume of 50 μl. A SpinPrepTM PCR Clean-Up kit (Novagen, Darmstadt, Germany) was used to purify the PCR products. Purified PCR products and pET-22b (+) (Novagen, Darmstadt, Germany) were digested with EcoRI and XhoI (NEB, Ipswich, Massachusetts, USA) and subsequently purified. The ligation was performed using T4 DNA ligase (NEB, Ipswich, Massachusetts, USA) and then transformed into E. coli DH5a. The recombinant plasmid was confirmed by sequencing. The recombinant plasmid was then transformed into the expression host E. coli BL21 (DE3) for later analysis.
3
Chromatin Crosslinking and ChIA-PET
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Chromatin was fixed with 3% paraformaldehyde followed by further cross-linking with 10 mM dimethyl adipimidate (DMA). Crosslinked chromatin was sheared into 300–500 bp DNA fragments by Bioruptor (Diagenode). Cut14-Pk and Rad21-Myc proteins were immunoprecipitated using mouse monoclonal anti-PK (SV5-Pk1, Serotec) or mouse monoclonal anti-Myc (9E10, Clontech), and protein G-coupled Dynabeads (Life Technologies). Immunoprecipitated chromatin was subjected to the ChIA-PET procedure20 . ChIP DNA was split into an equal amount and separately ligated to either A or B linkers, and mixed ligation products were further subjected to proximity ligation. The ligation product was digested by EcoP15I and purified using streptavidin-coupled magnetic beads (Invitrogen). Illumina sequencing adaptors were added to DNA fragments, which were then subjected to PCR amplification (Phusion PCR Master Mix; New England Biolabs). This PCR was performed in a total of 32 separate tubes for each ChIA-PET library. Assembled PCR products were sequenced on the Illumina HiSeq 2000 platform to obtain 100 bp single end reads.
4
HFIP-based Polymer Fabrication and Cell Culture Protocol
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1, 1, 1, 3, 3, 3 hexafluoro 2-propanol (HFIP) was provided by Shanghai Darui Finechemical (Shanghai, China). Poly(lactic-co-glycolic) acid (PLGA, molar ratio of PLA: PGA = 75:25) was provided by Jinan Daigang Biomaterial (Jinan, China). Competent cells, chemicals and kits were provided by Sangon Biotech (Shanghai, China). Restriction enzymes were provided by Thermo scientific (Waltham, MA, USA). Phusion PCR Master Mix and T4 ligase were provided by New England Biolabs (Beijing, China). Human bone mesenchymal stem cells (HBMSCs) were provided by Shanghai Institute of Biochemistry and Cell Biology (SIBCB, CAS, Shanghai, China). They are commercialized cell lines. Dimethyl sulfoxide (DMSO) was provided by Changshu Hong sheng Chemical Reagent (Suzhou, China). Paraformaldehyde (POM) and Cell Counting Kit -8 (CCK-8) were provided by Biyotime Institute of Biotechnology (Haimen, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were provided by Mesgen Company (Shanghai, China). Penicillin/streptomycin and trypsin were provided by Shanghai Yuanxiang Medical Equipment (Shanghai, China). A Milli-Q Plus 185 water purification system (Millipore, Burlington, MA, USA) was utilized to produce ultrapure water (18 MΩ cm).
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